Gene editing systems comprising an rna guide targeting lactate dehydrogenase a (ldha) and uses thereof

ABSTRACT

Provided herein are gene editing systems and/or compositions comprising RNA guides targeting LDHA for use in genetic editing of the LDHA gene. Also provide herein are methods of using the gene editing system for introducing edits to the LDHA gene and/or for treatment of primary hyperoxaluria (PH), and processes for characterizing the gene editing system.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/197,067, filed Jun. 4, 2021, U.S. Provisional Application No. 63/225,214, filed Jul. 23, 2021, U.S. Provisional Application No. 63/292,912, filed Dec. 22, 2021, and U.S. Provisional Application No. 63/300,743, filed Jan. 19, 2022, the contents of each of which are incorporated by reference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 3, 2022, is named 116928-0036-0003US00_SEQ.txt and is 381,734 bytes in size.

BACKGROUND

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) genes, collectively known as CRISPR-Cas or CRISPR/Cas systems, are adaptive immune systems in archaea and bacteria that defend particular species against foreign genetic elements.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the development of a system for genetic editing of a lactate dehydrogenase A (LDHA) gene. The system involves a Cas12i polypeptide such as a Cas12i2 polypeptide and an RNA guide mediating cleavage at a genetic site within the LDHA gene by the CRISPR nuclease polypeptide. As reported herein, the gene editing system disclosed herein has achieved successful editing of LDHA gene with high editing efficiency and accuracy.

Without being bound by theory, the gene editing system disclosed herein may exhibit one or more of the following advantageous features. Compared to SpCas9 and Cas12a, Cas12i effectors are smaller (1033 to 1093aa) which, in conjunction with their short mature crRNA (40-43 nt), is preferable in terms of delivery and cost of synthesis. Cas12i cleavage results in larger deletions compared to the small deletions and +1 insertions induced by Cas9 cleavage. Cas12i PAM sequences also differ from those of Cas9. Therefore, larger and different portions of genetic sites of interest can be disrupted with a Cas12i polypeptide and RNA guide compared to Cas9. Using an unbiased approach of tagmentation-based tag integration site sequencing (TTISS), more potential off-target sites with a higher number of unique integration events were identified for SpCas9 compared to Cas12i2. See WO/2021/202800. Therefore, Cas12i such as Cas12i2 may be more specific than Cas9.

Accordingly, provided herein are gene editing systems for editing LDHA gene, pharmaceutical compositions or kits comprising such, methods of using the gene editing systems to produce genetically modified cells, and the resultant cells thus produced. Also provided herein are uses of the gene editing systems disclosed herein, the pharmaceutical compositions and kits comprising such, and/or the genetically modified cells thus produced for treating primary hyperoxaluria (PH) in a subject.

In some aspects, the present disclosure features system for genetic editing of a hydroxyacid oxidase 1 (LDHA) gene, comprising (i) a Cas12i polypeptide or a first nucleic acid encoding the Cas12i polypeptide, and (ii) an RNA guide or a second nucleic acid encoding the RNA guide. The RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence.

In some embodiments, the Cas12i is a Cas12i2 polypeptide. In other embodiments, the Cas12i is a Cas12i4 polypeptide.

In some embodiments, the Cas12i polypeptide is a Cas12i2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 1166. In some instances, the Cas12i2 polypeptide may comprise one or more mutations relative to SEQ ID NO: 1166. In some examples, the one or more mutations in the Cas12i2 polypeptide are at positions D581, G624, F626, P868, I926, V1030, E1035, and/or S1046 of SEQ ID NO: 1166. In some examples, the one or more mutations are amino acid substitutions, which optionally is D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.

In one example, the Cas12i2 polypeptide comprises mutations at positions D581, D911, 1926, and V1030 (e.g., amino acid substitutions of D581R, D911R, I926R, and V1030G). In another example, the Cas12i2 polypeptide comprises mutations at positions D581, I926, and V1030 (e.g., amino acid substitutions of D581R, I926R, and V1030G). In yet another example, the Cas12i2 polypeptide comprises mutations at positions D581, I926, V1030, and S1046 (e.g., amino acid substitutions of D581R, I926R, V1030G, and S1046G). In still another example, the Cas12i2 polypeptide comprises mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G). In another example, the Cas12i2 polypeptide comprises mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G).

Exemplary Cas12i2 polypeptides for use in any of the gene editing systems disclosed herein may comprise the amino acid sequence of any one of SEQ ID NOs: 1167-1171. In one example, the exemplary Cas12i2 polypeptide for use in any of the gene editing systems disclosed herein comprises the amino acid sequence of SEQ ID NO: 1168. In another example, the exemplary Cas12i2 polypeptide for use in any of the gene editing systems disclosed herein comprises the amino acid sequence of SEQ ID NO: 1171.

In some embodiments, the gene editing system may comprise the first nucleic acid encoding the Cas12i polypeptide (e.g., the Cas12i2 polypeptide). In some instances, the first nucleic acid is located in a first vector (e.g., a viral vector such as an adeno-associated viral vector or AAV vector). In some instances, the first nucleic acid is a messenger RNA (mRNA). In some instances, the coding sequence for the Cas12i polypeptide is codon optimized.

In some embodiments, the target sequence may be within exon 1 or exon 2 of the LDHA gene. In some examples, the target sequence comprises 5′-TAGGACTTGGCAGATGAACT-3′ (SEQ ID NO: 1237), 5′-GATGACATCAACAAGAGCAA-3′ (SEQ ID NO: 1239), 5′-TTCATAGTGGATATCTTGAC-3′ (SEQ ID NO: 1245), 5′-TCATAGTGGATATCTTGACC-3′ (SEQ ID NO: 1248), or 5′-CATAGTGGATATCTTGACCT-3′ (SEQ ID NO: 1249). In some examples, the target sequence may comprise SEQ ID NO: 1248.

In some embodiments, the spacer sequence may be 20-30-nucleotide in length. In some examples, the spacer sequence is 20-nucleotide in length. In some examples, the spacer sequence comprises 5′-UAGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1269); 5′-GAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1270); 5′-UUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1271); 5′-UCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1272); or 5′-CAUAGUGGAUAUCUUGACCU-3′ (SEQ ID NO: 1273). In some examples, the spacer sequence may comprise SEQ ID NO: 1272.

In some embodiments, the RNA guide comprises the spacer and a direct repeat sequence. In some examples, the direct repeat sequence is 23-36-nucleotide in length. In one example, the direct repeat sequence is at least 90% identical to any one of SEQ ID NOs: 1-10 or a fragment thereof that is at least 23-nucleotide in length. In some specific examples, the direct repeat sequence is any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23-nucleotide in length. By way of non-limiting example, the direct repeat sequence is 5′-AGAAAUCCGUCUUUCAUUGACGG-3′ (SEQ ID NO: 10).

In specific examples, the RNA guide may comprise the nucleotide sequence of 5′-AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1214), 5′-AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1235), 5′-AGAAAUCCGUCUUUCAUUGACGGUUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1221), 5′-AGAAAUCCGUCUUUCAUUGACGGUCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1224), or 5′-AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU-3′ (SEQ ID NO: 1225). In one example, the RNA guide may comprise SEQ ID NO: 1224.

In some embodiments, the system may comprise the second nucleic acid encoding the RNA guide. In some examples, the nucleic acid encoding the RNA guide may be located in a viral vector. In some examples, the viral vector comprises the both the first nucleic acid encoding the Cas12i polypeptide (e.g., the Cas12i2 polypeptide) and the second nucleic acid encoding the RNA guide.

In some embodiments, any of the systems described herein may comprise the first nucleic acid encoding the Cas12i polypeptide (e.g., the Cas12i2 polypeptide), which is located in a first vector, and the second nucleic acid encoding the RNA guide, which is located on a second vector. In some examples, the first and/or second vector is a viral vector. In some specific examples, the first and second vectors are the same vector.

In some embodiments, any of the systems described herein may comprise one or more lipid nanoparticles (LNPs), which encompass the Cas12i polypeptide (e.g., the Cas12i2 polypeptide) or the first nucleic acid encoding the Cas12i polypeptide, the RNA guide or the second nucleic acid encoding the RNA guide, or both.

In some embodiments, the system described herein may comprise a LNP, which encompass the Cas12i polypeptide (e.g., the Cas12i2 polypeptide) or the first nucleic acid encoding the Cas12i polypeptide, and a viral vector comprising the second nucleic acid encoding the RNA guide. In some examples, the viral vector is an AAV vector. In other embodiments, the system described herein may comprise a LNP, which encompass the RNA guide or the second nucleic acid encoding the RNA guide, and a viral vector comprising the first nucleic acid encoding the Cas12i polypeptide. In some examples, the viral vector is an AAV vector.

In some aspects, the present disclosure also provides a pharmaceutical composition comprising any of the gene editing systems disclosed herein, and a kit comprising the components of the gene editing system.

In other aspects, the present disclosure also features a method for editing a lactate dehydrogenase A (LDHA) gene in a cell, the method comprising contacting a host cell with any of the systems disclosed herein to genetically edit the LDHA gene in the host cell. In some examples, the host cell is cultured in vitro. In other examples, the contacting step is performed by administering the system for editing the LDHA gene to a subject comprising the host cell.

Also within the scope of the present disclosure is a cell comprising a disrupted a lactate dehydrogenase A (LDHA) gene, which can be produced by contacting a host cell with the system disclosed herein genetically edit the LDHA gene in the host cell.

Still in other aspects, the present disclosure provides a method for treating primary hyperoxaluria (PH) in a subject. The method may comprise administering to a subject in need thereof any of the systems for editing a lactate dehydrogenase A (LDHA) gene or any of the cells disclosed herein.

In some embodiments, the subject may be a human patient having the PH. In some examples, the PH is PH1, PH2, or PH3. In a specific example, the PH is PH1.

Also provided herein is an RNA guide, comprising (i) a spacer sequence as disclosed herein that is specific to a target sequence in a lactate dehydrogenase A (LDHA) gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence; and (ii) a direct repeat sequence.

In some embodiments, the spacer may be 20-30-nucleotide in length. In some examples, the spacer is 20-nucleotide in length.

In some embodiments, the direct repeat sequence may be 23-36-nucleotide in length. In some examples, the direct repeat sequence is 23-nucleotide in length.

In some embodiments, the target sequence may be within exon 3 or exon 5 of the LDHA gene. In some examples, the target sequence comprises 5′-TAGGACTTGGCAGATGAACT-3′ (SEQ ID NO: 1237), 5′-GATGACATCAACAAGAGCAA-3′ (SEQ ID NO: 1239), 5′-TTCATAGTGGATATCTTGAC-3′ (SEQ ID NO: 1245), 5′-TCATAGTGGATATCTTGACC-3′ (SEQ ID NO: 1248), or 5′-CATAGTGGATATCTTGACCT-3′ (SEQ ID NO: 1249). In some examples, the target sequence may comprise SEQ ID NO: 1248.

In some embodiments, the spacer sequence may comprise 5′-AGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1269); 5′-GAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1270); 5′-UUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1271); 5′-UCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1272); or 5′-CAUAGUGGAUAUCUUGACCU-3 (SEQ ID NO: 1273). In some examples, the spacer sequence may comprise SEQ ID NO: 1272.

In some embodiments, the direct repeat sequence may be at least 90% identical to any one of SEQ ID NOs: 1-10 or a fragment thereof that is at least 23-nucleotide in length. In some examples, the direct repeat sequence is any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23-nucleotide in length. By way of non-limiting example, the direct repeat sequence is 5′-AGAAAUCCGUCUUUCAUUGACGG-3′ (SEQ ID NO: 10).

In some embodiments, the RNA guide may comprise the nucleotide sequence of 5′-AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1214), 5′-AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1235), 5′-AGAAAUCCGUCUUUCAUUGACGGUUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1221), 5′-AGAAAUCCGUCUUUCAUUGACGGUCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1224), or 5′-AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU-3′ (SEQ ID NO: 1225). In some examples, the RNA guide may comprise SEQ ID NO: 1224.

Also provided herein are any of the gene editing systems disclosed herein, pharmaceutical compositions or kits comprising such, or genetically modified cells generated by the gene editing system for use in treating PH in a subject, as well as uses of the gene editing systems disclosed herein, pharmaceutical compositions or kits comprising such, or genetically modified cells generated by the gene editing system for manufacturing a medicament for treatment of PH in a subject.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the ability of RNPs prepared with a Cas12i2 polypeptide and a crRNA to edit the LDHA gene in HEK293 cells. The darker grey bars represent target sequences with perfect homology to both rhesus macaque (Macaca mulatta) and crab-eating macaque (Macaca fascicularis) sequences.

FIG. 2 is a graph showing the ability of RNPs prepared with a Cas12i2 polypeptide and a crRNA to edit LDHA target sequences in HepG2 cells.

FIG. 3 is a graph showing the ability of RNPs prepared with a Cas12i2 polypeptide and a crRNA to edit LDHA target sequences in primary hepatocytes.

FIG. 4 is a graph showing knockdown of LDHA mRNA in primary human hepatocytes with a Cas12i2 polypeptide and an LDHA-targeting crRNA, E3T1 (SEQ ID NO: 1214).

FIG. 5A is a graph showing % indels induced by LDHA-targeting crRNAs and the variant Cas12i2 polypeptide of SEQ ID NO: 1168 or SEQ ID NO: 1171 in HepG2 cells. FIG. 5B shows the size (left) and start position (right) of indels induced in HepG2 cells by the variant Cas12i2 of SEQ ID NO: 1168 and the LDHA-targeting RNA guide of E5T9 (SEQ ID NO: 1224).

FIG. 6 is a graph showing % indels induced by chemically modified LDHA-targeting crRNAs of SEQ ID NO: 1267 and SEQ ID NO: 1268 and the variant Cas12i2 mRNA of SEQ ID NO: 1265 or SEQ ID NO: 1266.

FIG. 7A shows plots depicting tagmentation-based tag integration site sequencing (TTISS) reads for variant Cas12i2 of SEQ ID NO: 1168 and LDHA-targeting RNA guides E5T9 (SEQ ID NO: 1224), E3T1 (SEQ ID NO: 1214), E5T10 (SEQ ID NO: 1225), and EST1 (SEQ ID NO: 1221). The black wedge and centered number represent the fraction of on-target TTISS reads. Each gray wedge represents a unique off-target site identified by TTISS. The size of each gray wedge represents the fraction of TTISS reads mapping to a given off-target. FIG. 7B shows plots depicting two replicates of TTISS reads for variant Cas12i2 of SEQ ID NO: 1171 and LDHA-targeting RNA guides E5T9 (SEQ ID NO: 1224), E5T10 (SEQ ID NO: 1225), and E3T1 (SEQ ID NO: 1214). The black wedge and centered number represent the fraction of on-target TTISS reads. Each gray wedge represents a unique off-target site identified by TTISS. The size of each gray wedge represents the fraction of TTISS reads mapping to a given off-target.

FIG. 8 is a Western Blot showing knockdown of LDHA protein following electroporation of primary human hepatoyctes with variant Cas12i2 of SEQ ID NO: 1168 and RNA guides E3T1 (SEQ ID NO: 1214), E5T9 (SEQ ID NO: 1224), E5T1 (SEQ ID NO: 1221), or E5T10 (SEQ ID NO: 1225).

DETAILED DESCRIPTION

The present disclosure relates to a system for genetic editing of a lactate dehydrogenase A (LDHA) gene, which comprises (i) a Cas12i polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide; and (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence. Also provided in the present disclosure are a pharmaceutical composition or a kit comprising such system as well as uses thereof. Further disclosed herein are a method for editing a LDHA gene in a cell, a cell so produced that comprises a disrupted a LDHA gene, a method of treating primary hyperoxaluria (PH) in a subject, and an RNA guide that comprises (i) a spacer that is specific to a target sequence in a LDHA gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence; and (ii) a direct repeat sequence as well as uses thereof.

The Cas12i polypeptide for use in the gene editing system disclosed herein may be a Cas12i2 polypeptide, e.g., a wild-type Cas12i polypeptide or a variant thereof as those disclosed herein. In some examples, the Cas12i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 922 and comprises one or more mutations relative to SEQ ID NO: 922. In other examples, the Cas12i polypeptide may be a Cas12i4 polypeptide, which is also disclosed herein.

Definitions

The present disclosure will be described with respect to particular embodiments and with reference to certain Figures, but the disclosure is not limited thereto but only by the claims. Terms as set forth hereinafter are generally to be understood in their common sense unless indicated otherwise.

As used herein, the term “activity” refers to a biological activity. In some embodiments, activity includes enzymatic activity, e.g., catalytic ability of a Cas12i polypeptide. For example, activity can include nuclease activity.

As used herein the term “LDHA” refers to “lactate dehydrogenase A.” LDHA is an enzyme that catalyzes the inter-conversion of pyruvate and L-lactate with concomitant inter-conversion of NADH and NAD+. LDHA plays roles in development, as well as invasion and metastasis of cancer. Many cancers are characterized by higher LDHA levels than normal tissues. SEQ ID NO: 1172 as set forth herein provides an example of an LDHA gene sequence.

As used herein, the term “Cas12i polypeptide” (also referred to herein as Cas12i) refers to a polypeptide that binds to a target sequence on a target nucleic acid specified by an RNA guide, wherein the polypeptide has at least some amino acid sequence homology to a wild-type Cas12i polypeptide. In some embodiments, the Cas12i polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any one of SEQ ID NOs: 1-5 and 11-18 of U.S. Pat. No. 10,808,245, which is incorporated by reference for the subject matter and purpose referenced herein. In some embodiments, a Cas12i polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any one of SEQ ID NOs: 8, 2, 11, and 9 of the present application. In some embodiments, a Cas12i polypeptide of the disclosure is a Cas12i2 polypeptide as described in WO/2021/202800, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. In some embodiments, the Cas12i polypeptide cleaves a target nucleic acid (e.g., as a nick or a double strand break).

As used herein, the term “complex” refers to a grouping of two or more molecules. In some embodiments, the complex comprises a polypeptide and a nucleic acid molecule interacting with (e.g., binding to, coming into contact with, adhering to) one another. For example, the term “complex” can refer to a grouping of an RNA guide and a polypeptide (e.g., a Cas12i polypeptide). Alternatively, the term “complex” can refer to a grouping of an RNA guide, a polypeptide, and the complementary region of a target sequence. In another example, the term “complex” can refer to a grouping of an LDHA-targeting RNA guide and a Cas12i polypeptide.

As used herein, the term “protospacer adjacent motif” or “PAM” refers to a DNA sequence adjacent to a target sequence (e.g., an LDHA target sequence) to which a complex comprising an RNA guide (e.g., an LDHA-targeting RNA guide) and a Cas12i polypeptide binds. In a double-stranded DNA molecule, the strand containing the PAM motif is called the “PAM-strand” and the complementary strand is called the “non-PAM strand.” The RNA guide binds to a site in the non-PAM strand that is complementary to a target sequence disclosed herein. In some embodiments, the PAM strand is a coding (e.g., sense) strand. In other embodiments, the PAM strand is a non-coding (e.g., antisense strand). Since an RNA guide binds the non-PAM strand via base-pairing, the non-PAM strand is also known as the target strand, while the PAM strand is also known as the non-target strand.

As used herein, the term “target sequence” refers to a DNA fragment adjacent to a PAM motif (on the PAM strand). The complementary region of the target sequence is on the non-PAM strand. A target sequence may be immediately adjacent to the PAM motif. Alternatively, the target sequence and the PAM may be separately by a small sequence segment (e.g., up to 5 nucleotides, for example, up to 4, 3, 2, or 1 nucleotide). A target sequence may be located at the 3′ end of the PAM motif or at the 5′ end of the PAM motif, depending upon the CRISPR nuclease that recognizes the PAM motif, which is known in the art. For example, a target sequence is located at the 3′ end of a PAM motif for a Cas12i polypeptide (e.g., a Cas12i2 polypeptide such as those disclosed herein). In some embodiments, the target sequence is a sequence within an LDHA gene sequence, including, but not limited, to the sequence set forth in SEQ ID NO: 1172.

As used herein, the term “adjacent to” refers to a nucleotide or amino acid sequence in close proximity to another nucleotide or amino acid sequence. In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if no nucleotides separate the two sequences (i.e., immediately adjacent). In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if a small number of nucleotides separate the two sequences (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by up to 2 nucleotides, up to 5 nucleotides, up to 8 nucleotides, up to 10 nucleotides, up to 12 nucleotides, or up to 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by 2-5 nucleotides, 4-6 nucleotides, 4-8 nucleotides, 4-10 nucleotides, 6-8 nucleotides, 6-10 nucleotides, 6-12 nucleotides, 8-10 nucleotides, 8-12 nucleotides, 10-12 nucleotides, 10-15 nucleotides, or 12-15 nucleotides.

As used herein, the term “spacer” or “spacer sequence” is a portion in an RNA guide that is the RNA equivalent of the target sequence (a DNA sequence). The spacer contains a sequence capable of binding to the non-PAM strand via base-pairing at the site complementary to the target sequence (in the PAM strand). Such a spacer is also known as specific to the target sequence. In some instances, the spacer may be at least 75% identical to the target sequence (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%), except for the RNA-DNA sequence difference. In some instances, the spacer may be 100% identical to the target sequence except for the RNA-DNA sequence difference.

As used herein, the term “RNA guide” or “RNA guide sequence” refers to any RNA molecule or a modified RNA molecule that facilitates the targeting of a polypeptide (e.g., a Cas12i polypeptide) described herein to a target sequence (e.g., a sequence of an LDHA gene). For example, an RNA guide can be a molecule that is designed to include sequences that are complementary to a specific nucleic acid sequence (e.g., an LDHA nucleic acid sequence). An RNA guide may comprise a DNA targeting sequence (i.e., a spacer sequence) and a direct repeat (DR) sequence. In some instances, the RNA guide can be a modified RNA molecule comprising one or more deoxyribonucleotides, for example, in a DNA-binding sequence contained in the RNA guide, which binds a sequence complementary to the target sequence. In some examples, the DNA-binding sequence may contain a DNA sequence or a DNA/RNA hybrid sequence. The terms CRISPR RNA (crRNA), pre-crRNA and mature crRNA are also used herein to refer to an RNA guide.

As used herein, the term “complementary” refers to a first polynucleotide (e.g., a spacer sequence of an RNA guide) that has a certain level of complementarity to a second polynucleotide (e.g., the complementary sequence of a target sequence) such that the first and second polynucleotides can form a double-stranded complex via base-pairing to permit an effector polypeptide that is complexed with the first polynucleotide to act on (e.g., cleave) the second polynucleotide. In some embodiments, the first polynucleotide may be substantially complementary to the second polynucleotide, i.e., having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the second polynucleotide. In some embodiments, the first polynucleotide is completely complementary to the second polynucleotide, i.e., having 100% complementarity to the second polynucleotide.

The “percent identity” (a.k.a., sequence identity) of two nucleic acids or of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength-12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the present disclosure. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

As used herein, the term “edit” refers to one or more modifications introduced into a target nucleic acid, e.g., within the LDHA gene. The edit can be one or more substitutions, one or more insertions, one or more deletions, or a combination thereof. As used herein, the term “substitution” refers to a replacement of a nucleotide or nucleotides with a different nucleotide or nucleotides, relative to a reference sequence. As used herein, the term “insertion” refers to a gain of a nucleotide or nucleotides in a nucleic acid sequence, relative to a reference sequence. As used herein, the term “deletion” refers to a loss of a nucleotide or nucleotides in a nucleic acid sequence, relative to a reference sequence.

No particular process is implied in how to make a sequence comprising a deletion. For instance, a sequence comprising a deletion can be synthesized directly from individual nucleotides. In other embodiments, a deletion is made by providing and then altering a reference sequence. The nucleic acid sequence can be in a genome of an organism. The nucleic acid sequence can be in a cell. The nucleic acid sequence can be a DNA sequence. The deletion can be a frameshift mutation or a non-frameshift mutation. A deletion described herein refers to a deletion of up to several kilobases.

As used herein, the terms “upstream” and “downstream” refer to relative positions within a single nucleic acid (e.g., DNA) sequence in a nucleic acid molecule. “Upstream” and “downstream” relate to the 5′ to 3′ direction, respectively, in which RNA transcription occurs. A first sequence is upstream of a second sequence when the 3′ end of the first sequence occurs before the 5′ end of the second sequence. A first sequence is downstream of a second sequence when the 5′ end of the first sequence occurs after the 3′ end of the second sequence. In some embodiments, the 5′-NTTN-3′ or 5′-TTN-3′ sequence is upstream of an indel described herein, and a Cas12i-induced indel is downstream of the 5′-NTTN-3′ or 5′-TTN-3′ sequence.

I. Gene Editing Systems

In some aspects, the present disclosure provides gene editing systems comprising an RNA guide targeting an LDHA gene or a portion of the LDHA gene. Such a gene editing system can be used to edit the LDHA target gene, e.g., to disrupt the LDHA gene.

Lactate dehydrogenase (LDH) is an enzyme found in nearly every cell that regulates both the homeostasis of lactate and pyruvate, and of glyoxylate and oxalate metabolism. LDH is comprised of 4 polypeptides that form a tetramer. Five isozymes of LDH differing in their subunit composition and tissue distribution have been identified. The two most common forms of LDH are the muscle (M) form encoded by the LDHA gene, and the heart (H) form encoded by LDHB gene. In the perioxisome of liver cells, LDH is the key enzyme responsible for converting glyoxalate to oxalate which is then secreted into the plasma and excreted by the kidneys. As LDH is key in the final step of oxalate production, reduction of LDHA can reduce hepatic LDH and prevent calcium oxalate crystal deposition.

In some embodiments, the RNA guide is comprised of a direct repeat component and a spacer component. In some embodiments, the RNA guide binds a Cas12i polypeptide. In some embodiments, the spacer component is specific to an LDHA target sequence, wherein the LDHA target sequence is adjacent to a 5′-NTTN-3′ or 5′-TTN-3′ PAM sequence as described herein. In the case of a double-stranded target, the RNA guide binds to a first strand of the target (i.e., the non-PAM strand) and a PAM sequence as described herein is present in the second, complementary strand (i.e., the PAM strand).

In some embodiments, the present disclosure described herein comprises compositions comprising a complex, wherein the complex comprises an RNA guide targeting LDHA. In some embodiments, the present disclosure comprises a complex comprising an RNA guide and a Cas12i polypeptide. In some embodiments, the RNA guide and the Cas12i polypeptide bind to each other in a molar ratio of about 1:1. In some embodiments, a complex comprising an RNA guide and a Cas12i polypeptide binds to an LDHA target sequence. In some embodiments, a complex comprising an RNA guide targeting LDHA and a Cas12i polypeptide binds to an LDHA target sequence at a molar ratio of about 1:1. In some embodiments, the complex comprises enzymatic activity, such as nuclease activity, that can cleave the LDHA target sequence. The RNA guide, the Cas12i polypeptide, and the LDHA target sequence, either alone or together, do not naturally occur. In some embodiments, the RNA guide in the complex comprises a direct repeat and/or a spacer sequence described herein. In some embodiments, the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229. In some embodiments, the RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229.

In some embodiments, the present disclosure described herein comprises compositions comprising an RNA guide as described herein and/or an RNA encoding a Cas12i polypeptide as described herein. In some embodiments, the RNA guide and the RNA encoding a Cas12i polypeptide are comprised together within the same composition. In some embodiments, the RNA guide and the RNA encoding a Cas12i polypeptide are comprised within separate compositions. In some embodiments, the RNA guide comprises a direct repeat and/or a spacer sequence described herein. In some embodiments, the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229. In some embodiments, the RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229.

Use of the gene editing systems disclosed herein has advantages over those of other known nuclease systems. Cas12i polypeptides are smaller than other nucleases. For example, Cas12i2 is 1,054 amino acids in length, whereas S. pyogenes Cas9 (SpCas9) is 1,368 amino acids in length, S. thermophilus Cas9 (StCas9) is 1,128 amino acids in length, FnCpf1 is 1,300 amino acids in length, AsCpf1 is 1,307 amino acids in length, and LbCpf1 is 1,246 amino acids in length. Cas12i RNA guides, which do not require a trans-activating CRISPR RNA (tracrRNA), are also smaller than Cas9 RNA guides. The smaller Cas12i polypeptide and RNA guide sizes are beneficial for delivery. Compositions comprising a Cas12i polypeptide also demonstrate decreased off-target activity compared to compositions comprising an SpCas9 polypeptide. See WO/2021/202800, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. Furthermore, indels induced by compositions comprising a Cas12i polypeptide differ from indels induced by compositions comprising an SpCas9 polypeptide. For example, SpCas9 polypeptides primarily induce insertions and deletions of 1 nucleotide in length. However, Cas12i polypeptides induce larger deletions, which can be beneficial in disrupting a larger portion of a gene such as LDHA.

Also provided herein is a system for genetic editing of an LDHA gene, which comprises (i) a Cas12i polypeptide (e.g., a Cas12i2 polypeptide) or a first nucleic acid encoding the Cas12i polypeptide (e.g., a Cas12i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1166, which may comprise one or more mutations relative to SEQ ID NO: 1166); and (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene (e.g., within exon 3 or exon 5 of the LDHA gene), the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′ (5′-NTTN-3′), which is located 5′ to the target sequence.

A. RNA Guides

In some embodiments, the gene editing system described herein comprises an RNA guide targeting an LDHA gene, e.g., targeting exon 3 or exon 5 of the LDHA gene. In some embodiments, the gene editing system described herein comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) RNA guides targeting LDHA.

The RNA guide may direct the Cas12i polypeptide contained in the gene editing system as described herein to an LDHA target sequence. Two or more RNA guides may direct two or more separate Cas12i polypeptides (e.g., Cas12i polypeptides having the same or different sequence) as described herein to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) LDHA target sequences.

Those skilled in the art reading the below examples of particular kinds of RNA guides will understand that, in some embodiments, an RNA guide is LDHA target-specific. That is, in some embodiments, an RNA guide binds specifically to one or more LDHA target sequences (e.g., within a cell) and not to non-targeted sequences (e.g., non-specific DNA or random sequences within the same cell).

In some embodiments, the RNA guide comprises a spacer sequence followed by a direct repeat sequence, referring to the sequences in the 5′ to 3′ direction. In some embodiments, the RNA guide comprises a first direct repeat sequence followed by a spacer sequence and a second direct repeat sequence, referring to the sequences in the 5′ to 3′ direction. In some embodiments, the first and second direct repeats of such an RNA guide are identical. In some embodiments, the first and second direct repeats of such an RNA guide are different.

In some embodiments, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present within the same RNA molecule. In some embodiments, the spacer and direct repeat sequences are linked directly to one another. In some embodiments, a short linker is present between the spacer and direct repeat sequences, e.g., an RNA linker of 1, 2, or 3 nucleotides in length. In some embodiments, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present in separate molecules, which are joined to one another by base pairing interactions.

Additional information regarding exemplary direct repeat and spacer components of RNA guides is provided as follows.

(i). Direct Repeat

In some embodiments, the RNA guide comprises a direct repeat sequence. In some embodiments, the direct repeat sequence of the RNA guide has a length of between 12-100, 13-75, 14-50, or 15-40 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).

In some embodiments, the direct repeat sequence is a sequence of Table 1 or a portion of a sequence of Table 1. The direct repeat sequence can comprise nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 1 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 2 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 3 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 4 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 5 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 6 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 7 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 8 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 9 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 10 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 11 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 12 through nucleotide 34 of SEQ ID NO: 9. In some embodiments, the direct repeat sequence is set forth in SEQ ID NO: 10. In some embodiments, the direct repeat sequence comprises a portion of the sequence set forth in SEQ ID NO: 10.

In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 1 or a portion of a sequence of Table 1. The direct repeat sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 1 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 2 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 3 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 4 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 5 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 6 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 7 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 8 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 9 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 10 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 11 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 12 through nucleotide 34 of SEQ ID NO: 9. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to SEQ ID NO: 10. In some embodiments, the direct repeat sequence has at least 90% identity to a portion of the sequence set forth in SEQ ID NO: 10.

In some embodiments, compositions comprising a Cas12i2 polypeptide and an RNA guide comprising the direct repeat of SEQ ID NO: 10 and a spacer length of 20 nucleotides are capable of introducing indels into an LDHA target sequence. See, e.g., Example 1, where indels were measured at seventeen LDHA target sequences following delivery of an RNA guide and a Cas12i2 polypeptide of SEQ ID NO: 1168 to HEK293T cells by RNP; Example 2, where indels were measured at four LDHA target sequences following delivery of an RNA guide and a Cas12i2 polypeptide of SEQ ID NO: 1168 to HepG2 cells by RNP; and Example 3, where indels were measured at three LDHA target sequences following delivery of an RNA guide and a Cas12i2 polypeptide of SEQ ID NO: 1168 primary hepatocytes by RNP.

In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1-10. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1-10.

TABLE 1 Cas12i2 Direct Repeat Sequences Sequence Identifier Direct Repeat Sequence SEQ ID NO: 1 GUUGCAAAACCCAAGAAAUCCGUCUUUCAUUGACGG SEQ ID NO: 2 AAUAGCGGCCCUAAGAAAUCCGUCUUUCAUUGACGG SEQ ID NO: 3 AUUGGAACUGGCGAGAAAUCCGUCUUUCAUUGACGG SEQ ID NO: 4 CCAGCAACACCUAAGAAAUCCGUCUUUCAUUGACGG SEQ ID NO: 5 CGGCGCUCGAAUAGGAAAUCCGUCUUUCAUUGACGG SEQ ID NO: 6 GUGGCAACACCUAAGAAAUCCGUCUUUCAUUGACGG SEQ ID NO: 7 GUUGCAACACCUAAGAAAUCCGUCUUUCAUUGACGG SEQ ID NO: 8 GUUGCAAUGCCUAAGAAAUCCGUCUUUCAUUGACGG SEQ ID NO: 9 GCAACACCUAAGAAAUCCGUCUUUCAUUGACGGG SEQ ID NO: 10 AGAAAUCCGUCUUUCAUUGACGG

In some embodiments, the direct repeat sequence is a sequence of Table 2 or a portion of a sequence of Table 2. The direct repeat sequence can comprise nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs:

1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.

In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2 or a portion of a sequence of Table 2. The direct repeat sequence can have at least 95% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.

In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2 or a portion of a sequence of Table 2. The direct repeat sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.

In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.

In some embodiments, the direct repeat sequence is at least 90% identical to SEQ ID NO: 1200 or a portion of SEQ ID NO: 1200. In some embodiments, the direct repeat sequence is at least 95% identical to SEQ ID NO: 1200 or a portion of SEQ ID NO: 1200. In some embodiments, the direct repeat sequence is 100% identical to SEQ ID NO: 1200 or a portion of SEQ ID NO: 1200.

TABLE 2 Cas12i4 Direct Repeat Sequences Sequence Identifier Direct Repeat Sequence SEQ ID NO: UCUCAACGAUAGUCAGACAUGUGUCCUCAGUGACAC 1182 SEQ ID NO: UUUUAACAACACUCAGGCAUGUGUCCACAGUGACAC 1183 SEQ ID NO: UUGAACGGAUACUCAGACAUGUGUUUCCAGUGACAC 1184 SEQ ID NO: UGCCCUCAAUAGUCAGAUGUGUGUCCACAGUGACAC 1185 SEQ ID NO: UCUCAAUGAUACUUAGAUACGUGUCCUCAGUGACAC 1186 SEQ ID NO: UCUCAAUGAUACUCAGACAUGUGUCCCCAGUGACAC 1187 SEQ ID NO: UCUCAAUGAUACUAAGACAUGUGUCCUCAGUGACAC 1188 SEQ ID NO: UCUCAACUAUACUCAGACAUGUGUCCUCAGUGACAC 1189 SEQ ID NO: UCUCAACGAUACUCAGACAUGUGUCCUCAGUGACAC 1190 SEQ ID NO: UCUCAACGAUACUAAGAUAUGUGUCCUCAGCGACAC 1191 SEQ ID NO: UCUCAACGAUACUAAGAUAUGUGUCCCCAGUGACAC 1192 SEQ ID NO: UCUCAACGAUACUAAGAUAUGUGUCCACAGUGACAC 1193 SEQ ID NO: UCUCAACAAUACUCAGACAUGUGUCCCCAGUGACAC 1194 SEQ ID NO: UCUCAACAAUACUAAGGCAUGUGUCCCCAGUGACCC 1195 SEQ ID NO: UCUCAAAGAUACUCAGACACGUGUCCCCAGUGACAC 1196 SEQ ID NO: UCUCAAAAAUACUCAGACAUGUGUCCUCAGUGACAC 1197 SEQ ID NO: GCGAAACAACAGUCAGACAUGUGUCCCCAGUGACAC 1198 SEQ ID NO: CCUCAACGAUAUUAAGACAUGUGUCCGCAGUGACAC 1199 SEQ ID NO: AGACAUGUGUCCUCAGUGACAC 1200

In some embodiments, the direct repeat sequence is a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1205-1207. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1205-1207. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1205-1207.

TABLE 3 Cas12il Direct Repeat Sequences Sequence Direct Repeat Identifier Sequence SEQ ID NO: 1205 GUUGGAAUGACUAAUUUUUGUGC CCACCGUUGGCAC SEQ ID NO: 1206 AAUUUUUGUGCCCAUCGUUGGCAC SEQ ID NO: 1207 AUUUUUGUGCCCAUCGUUGGCAC

In some embodiments, the direct repeat sequence is a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1208-1210. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1208-1210. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1208-1210.

TABLE 4 Cas12i3 Direct Repeat Sequences Sequence Direct Repeat Identifier Sequence SEQ ID NO: 1208 CUAGCAAUGACCUAAUAGUGUG UCCUUAGUUGACAU SEQ ID NO: 1209 CCUACAAUACCUAAGAAAUCCG UCCUAAGUUGACGG SEQ ID NO: 1210 AUAGUGUGUCCUUAGUUGACAU

In some embodiments, a direct repeat sequence described herein comprises a uracil (U). In some embodiments, a direct repeat sequence described herein comprises a thymine (T). In some embodiments, a direct repeat sequence according to Tables 1-4 comprises a sequence comprising a thymine in one or more places indicated as uracil in Tables 1-4.

(ii). Spacer Sequence

In some embodiments, the RNA guide comprises a DNA targeting or spacer sequence. In some embodiments, the spacer sequence of the RNA guide has a length of between 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and is complementary to a non-PAM strand sequence). In some embodiments, the spacer sequence is designed to be complementary to a specific DNA strand, e.g., of a genomic locus.

In some embodiments, the RNA guide spacer sequence is substantially identical to a complementary strand of a target sequence. In some embodiments, the RNA guide comprises a sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a complementary strand of a reference nucleic acid sequence, e.g., target sequence. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.

In some embodiments, the RNA guide comprises a spacer sequence that has a length of between 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a region on the non-PAM strand that is complementary to the target sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence. In some embodiments, the RNA guide comprises a sequence, e.g., RNA sequence, that is a length of up to 50 and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a region on the non-PAM strand that is complementary to the target. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence.

In some embodiments, the spacer sequence is a sequence of Table 5 or a portion of a sequence of Table 5. It should be understood that an indication of SEQ ID NOs: 588-1164 should be considered as equivalent to a listing of SEQ ID NOs: 588-1164, with each of the intervening numbers present in the listing, i.e., 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, and 1164.

The spacer sequence can comprise nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 588-1164.

In some embodiments, the spacer sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 5 or a portion of a sequence of Table 5. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 30 of any one of 588-1164.

TABLE 5 Target and Spacer Sequences SEQ SEQ ID ID LDHA Strand PAM* NO Target Sequence NO Spacer Sequence LDHA_exon1 + ATTC 11 CGGATCTCATTGCCAC 588 CGGAUCUCAUUGCCACG GCGCCCCCGACGAC CGCCCCCGACGAC LDHA_exon1 + ATTG 12 CCACGCGCCCCCGACG 589 CCACGCGCCCCCGACGA ACCGCCCGACGTGC CCGCCCGACGUGC LDHA_exon1 + ATTC 13 CCGGTACGGTAGGGCC 590 CCGGUACGGUAGGGCCC CTGCGCGCACGGCG UGCGCGCACGGCG LDHA_exon2 + CTTG 14 CTGTAGGAGCCGGAGT 591 CUGUAGGAGCCGGAGUA AGCTCAGAGTGATC GCUCAGAGUGAUC LDHA_exon2 + OTTA 15 CACCCAAACGTCGATA 592 CACCCAAACGUCGAUAU TTCCTTTTCCACGC UCCUUUUCCACGC LDHA_exon2 + GTTA 16 ATAAACCGCGATGGGT 593 AUAAACCGCGAUGGGUG GAACCCTCAGGAGG AACCCUCAGGAGG LDHA_exon2 + CTTG 17 GGGTTAATAAACCGCG 594 GGGUUAAUAAACCGCGA ATGGGTGAACCCTC UGGGUGAACCCUC LDHA_exon2 + TTTA 18 CTTGAGAAGCCTGGCT 595 CUUGAGAAGCCUGGCUG GTGTCCTTGCTGTA UGUCCUUGCUGUA LDHA_exon2 + GTTT 19 ACTTGAGAAGCCTGGC 596 ACUUGAGAAGCCUGGCU TGTGTCCTTGCTGT GUGUCCUUGCUGU LDHA_exon2 + TTTC 20 TGCACGTATCTCTGGT 597 UGCACGUAUCUCUGGUG GTTTACTTGAGAAG UUUACUUGAGAAG LDHA_exon2 + TTTT 21 CTGCACGTATCTCTGG 598 CUGCACGUAUCUCUGGU TGTTTACTTGAGAA GUUUACUUGAGAA LDHA_exon2 + GTTA 22 ATGGCTTTTCTGCACG 599 AUGGCUUUUCUGCACGU TATCTCTGGTGTTT AUCUCUGGUGUUU LDHA_exon2 + ATTC 23 CTTTTCCACGCTAAGG 600 CUUUUCCACGCUAAGGU TATGGGCCTTCACT AUGGGCCUUCACU LDHA_exon2 + TTTG 24 TGGCAGTTAATGGCTT 601 UGGCAGUUAAUGGCUUU TTCTGCACGTATCT UCUGCACGUAUCU LDHA_exon2 + GTTT 25 GTGGCAGTTAATGGCT 602 GUGGCAGUUAAUGGCUU TTTCTGCACGTATC UUCUGCACGUAUC LDHA_exon2 + CTTG 26 AGCTTTGTGGCAGTTA 603 AGCUUUGUGGCAGUUAA ATGGCTTTTCTGCA UGGCUUUUCUGCA LDHA_exon2 + CTTG 27 GGCTTGAGCTTTGTGG 604 GGCUUGAGCUUUGUGGC CAGTTAATGGCTTT AGUUAAUGGCUUU LDHA_exon2 + TTTC 28 CGAGCGGGAAGGAGAG 605 CGAGCGGGAAGGAGAGC CCACAAAGCGCGCA CACAAAGCGCGCA LDHA_exon2 + CTTG 29 AGAAGCCTGGCTGTGT 606 AGAAGCCUGGCUGUGUC CCTTGCTGTAGGAG CUUGCUGUAGGAG LDHA_exon2 + CTTT 30 TCTGCACGTATCTCTG 607 UCUGCACGUAUCUCUGG GTGTTTACTTGAGA UGUUUACUUGAGA LDHA_exon2 + CTTG 31 TCTGAGGAAAGGCCAG 608 UCUGAGGAAAGGCCAGC CCCCACTTGGGGTT CCCACUUGGGGUU LDHA_exon2 + TTTT 32 CCACGCTAAGGTATGG 609 CCACGCUAAGGUAUGGG GCCTTCACTCTTCA CCUUCACUCUUCA LDHA_exon2 + TTTC 33 CGCCCACCTTTCCGAG 610 CGCCCACCUUUCCGAGC CGGGAAGGAGAGCC GGGAAGGAGAGCC LDHA_exon2 − CTTC 34 CCGCTCGGAAAGGTGG 611 CCGCUCGGAAAGGUGGG GCGGAAATCAGACT CGGAAAUCAGACU LDHA_exon2 − TTTG 35 TGGCTCTCCTTCCCGC 612 UGGCUCUCCUUCCCGCU TCGGAAAGGTGGGC CGGAAAGGUGGGC LDHA_exon2 − CTTT 36 GTGGCTCTCCTTCCCG 613 GUGGCUCUCCUUCCCGC CTCGGAAAGGTGGG UCGGAAAGGUGGG LDHA_exon2 − ATTA 37 ACTGCCACAAAGCTCA 614 ACUGCCACAAAGCUCAA AGCCCAAGGCACAG GCCCAAGGCACAG LDHA_exon2 − CTTC 38 TCAAGTAAACACCAGA 615 UCAAGUAAACACCAGAG GATACGTGCAGAAA AUACGUGCAGAAA LDHA_exon2 − TTTC 39 CTCAGACAAGATCACT 616 CUCAGACAAGAUCACUC CTGAGCTACTCCGG UGAGCUACUCCGG LDHA_exon2 − CTTT 40 CCTCAGACAAGATCAC 617 CCUCAGACAAGAUCACU TCTGAGCTACTCCG CUGAGCUACUCCG LDHA_exon2 − ATTA 41 ACCCCAAGTGGGGCTG 618 ACCCCAAGUGGGGCUGG GCCTTTCCTCAGAC CCUUUCCUCAGAC LDHA_exon2 − TTTA 42 TTAACCCCAAGTGGGG 619 UUAACCCCAAGUGGGGC CTGGCCTTTCCTCA UGGCCUUUCCUCA LDHA_exon2 − GTTT 43 ATTAACCCCAAGTGGG 620 AUUAACCCCAAGUGGGG GCTGGCCTTTCCTC CUGGCCUUUCCUC LDHA_exon2 − GTTC 44 ACCCATCGCGGTTTAT 621 ACCCAUCGCGGUUUAUU TAACCCCAAGTGGG AACCCCAAGUGGG LDHA_exon2 − TTTG 45 GGTGTAAGTATAGCCT 622 GGUGUAAGUAUAGCCUC CCTGAGGGTTCACC CUGAGGGUUCACC LDHA_exon2 − GTTT 46 GGGTGTAAGTATAGCC 623 GGGUGUAAGUAUAGCCU TCCTGAGGGTTCAC CCUGAGGGUUCAC LDHA_exon2 − CTTA 47 GCGTGGAAAAGGAATA 624 GCGUGGAAAAGGAAUAU TCGACGTTTGGGTG CGACGUUUGGGUG LDHA_exon2 + TTTC 48 CACGCTAAGGTATGGG 625 CACGCUAAGGUAUGGGC CCTTCACTCTTCAC CUUCACUCUUCAC LDHA_exon2 + GTTT 49 TCCACGCTAAGGTATG 626 UCCACGCUAAGGUAUGG GGCCTTCACTCTTC GCCUUCACUCUUC LDHA_exon2 + ATTT 50 CCGCCCACCTTTCCGA 627 CCGCCCACCUUUCCGAG GCGGGAAGGAGAGC CGGGAAGGAGAGC LDHA_exon2 + GTTT 51 CCGAGCGGGAAGGAGA 628 CCGAGCGGGAAGGAGAG GCCACAAAGCGCGC CCACAAAGCGCGC LDHA_exon2 + ATTA 52 GTCTGATTTCCGCCCA 629 GUCUGAUUUCCGCCCAC CCTTTCCGAGCGGG CUUUCCGAGCGGG LDHA_exon2 + CTTC 53 ACAGACCCTGTCATTA 630 ACAGACCCUGUCAUUAG GGCCT GCCU LDHA_exon2 + CTTC 54 ACTCTTCACAGACCCT 631 ACUCUUCACAGACCCUG GTCATTAGGCCT UCAUUAGGCCU LDHA_exon3 − ATTT 55 AGTGTCACTACAGCTT 632 AGUGUCACUACAGCUUC CTTTAATGTTTATT UUUAAUGUUUAUU LDHA_exon3 + GTTG 56 TTGGGGTTGGTGCTGT 633 UUGGGGUUGGUGCUGUU TGGCATGGCCTGTG GGCAUGGCCUGUG LDHA_exon3 + CTTC 57 TAAAGGAAGAACAGAC 634 UAAAGGAAGAACAGACC CCCCCAGAATAAGA CCCCAGAAUAAGA LDHA_exon3 + TTTA 58 TAATCTTCTAAAGGAA 635 UAAUCUUCUAAAGGAAG GAACAGACCCCCCA AACAGACCCCCCA LDHA_exon3 + ATTT 59 ATAATCTTCTAAAGGA 636 AUAAUCUUCUAAAGGAA AGAACAGACCCCCC GAACAGACCCCCC LDHA_exon3 + GTTC 60 CAAGTCCAATATGGCA 637 CAAGUCCAAUAUGGCAA ACTCTAAAGGATCA CUCUAAAGGAUCA LDHA_exon3 + TTTG 61 GTTCCAAGTCCAATAT 638 GUUCCAAGUCCAAUAUG GGCAACTCTAAAGG GCAACUCUAAAGG LDHA_exon3 + TTTT 62 GGTTCCAAGTCCAATA 639 GGUUCCAAGUCCAAUAU TGGCAACTCTAAAG GGCAACUCUAAAG LDHA_exon3 + CTTT 63 TGGTTCCAAGTCCAAT 640 UGGUUCCAAGUCCAAUA ATGGCAACTCTAAA UGGCAACUCUAAA LDHA_exon3 + ATTC 64 CTTTTGGTTCCAAGTC 641 CUUUUGGUUCCAAGUCC CAATATGGCAACTC AAUAUGGCAACUC LDHA_exon3 + TTTC 65 CTCCTATAGATTCCTT 642 CUCCUAUAGAUUCCUUU TTGGTTCCAAGTCC UGGUUCCAAGUCC LDHA_exon3 + TTTT 66 CCTCCTATAGATTCCT 643 CCUCCUAUAGAUUCCUU TTTGGTTCCAAGTC UUGGUUCCAAGUC LDHA_exon3 + TTTT 67 TCCTCCTATAGATTCC 644 UCCUCCUAUAGAUUCCU TTTTGGTTCCAAGT UUUGGUUCCAAGU LDHA_exon3 + GTTT 68 TTCCTCCTATAGATTC 645 UUCCUCCUAUAGAUUCC CTTTTGGTTCCAAG UUUUGGUUCCAAG LDHA_exon3 + ATTA 69 AAGAAGCTGTAGTGAC 646 AAGAAGCUGUAGUGACA ACTAAATGTTTTTC CUAAAUGUUUUUC LDHA_exon3 + GTTG 70 GGGTTGGTGCTGTTGG 647 GGGUUGGUGCUGUUGGC CATGGCCTGTGCCA AUGGCCUGUGCCA LDHA_exon3 + GTTG 71 GTGCTGTTGGCATGGC 648 GUGCUGUUGGCAUGGCC CTGTGCCATCAGTA UGUGCCAUCAGUA LDHA_exon3 + ATTA 72 CAGTTGTTGGGGTTGG 649 CAGUUGUUGGGGUUGGU TGCTGTTGGCATGG GCUGUUGGCAUGG LDHA_exon3 + CTTA 73 ATGAAGGTAAGTGAGA 650 AUGAAGGUAAGUGAGAG GTCTACCACACTGG UCUACCACACUGG LDHA_exon3 − GTTG 74 GAACCAAAAGGAATCT 651 GAACCAAAAGGAAUCUA ATAGGAGGAAAAAC UAGGAGGAAAAAC LDHA_exon3 − ATTG 75 GACTTGGAACCAAAAG 652 GACUUGGAACCAAAAGG GAATCTATAGGAGG AAUCUAUAGGAGG LDHA_exon3 − GTTG 76 CCATATTGGACTTGGA 653 CCAUAUUGGACUUGGAA ACCAAAAGGAATCT CCAAAAGGAAUCU LDHA_exon3 + GTTG 77 GCATGGCCTGTGCCAT 654 GCAUGGCCUGUGCCAUC CAGTATCTTAATGA AGUAUCUUAAUGA LDHA_exon3 − CTTT 78 AGAGTTGCCATATTGG 655 AGAGUUGCCAUAUUGGA ACTTGGAACCAAAA CUUGGAACCAAAA LDHA_exon3 − ATTA 79 TAAATCAGCTGATCCT 656 UAAAUCAGCUGAUCCUU TTAGAGTTGCCATA UAGAGUUGCCAUA LDHA_exon3 − TTTA 80 GAAGATTATAAATCAG 657 GAAGAUUAUAAAUCAGC CTGATCCTTTAGAG UGAUCCUUUAGAG LDHA_exon3 − CTTT 81 AGAAGATTATAAATCA 658 AGAAGAUUAUAAAUCAG GCTGATCCTTTAGA CUGAUCCUUUAGA LDHA_exon3 − TTTA 82 GAGTTGCCATATTGGA 659 GAGUUGCCAUAUUGGAC CTTGGAACCAAAAG UUGGAACCAAAAG LDHA_exon3 − GTTC 83 TTCCTTTAGAAGATTA 660 UUCCUUUAGAAGAUUAU TAAATCAGCTGATC AAAUCAGCUGAUC LDHA_exon3 − ATTO 84 TGGGGGGTCTGTTCTT 661 UGGGGGGUCUGUUCUUC CCTTTAGAAGATTA CUUUAGAAGAUUA LDHA_exon3 − CTTA 85 TTCTGGGGGGTCTGTT 662 UUCUGGGGGGUCUGUUC CTTCCTTTAGAAGA UUCCUUUAGAAGA LDHA_exon3 − ATTA 86 AGATACTGATGGCACA 663 AGAUACUGAUGGCACAG GGCCATGCCAACAG GCCAUGCCAACAG LDHA_exon3 − CTTC 87 ATTAAGATACTGATGG 664 AUUAAGAUACUGAUGGC CACAGGCCATGCCA ACAGGCCAUGCCA LDHA_exon3 − CTTA 88 CCTTCATTAAGATACT 665 CCUUCAUUAAGAUACUG GATGGCACAGGCCA AUGGCACAGGCCA LDHA_exon3 − CTTC 89 CAGTGTGGTAGACTCT 666 CAGUGUGGUAGACUCUC CACTTACCTTCATT ACUUACCUUCAUU LDHA_exon3 − CTTC 90 CTTTAGAAGATTATAA 667 CUUUAGAAGAUUAUAAA ATCAGCTGATCCTT UCAGCUGAUCCUU LDHA_exon3 − TTTA 91 GTGTCACTACAGCTTC 668 GUGUCACUACAGCUUCU TTTAATGTTTATT UUAAUGUUUAUU LDHA_exon4 − GTTC 92 TAAGGAAAAGGCTGCC 669 UAAGGAAAAGGCUGCCA ATGTTGGAGATCCA UGUUGGAGAUCCA LDHA_exon4 − GTTG 93 GAGATCCATCATCTCT 670 GAGAUCCAUCAUCUCUC CCCTTCAATTTGTC CCUUCAAUUUGUC LDHA_exon4 − CTTC 94 AATTTGTCTTCGATGA 671 AAUUUGUCUUCGAUGAC CATCAACAAGAGCA AUCAACAAGAGCA LDHA_exon4 − GTTC 95 ATCTGCCAAGTCCTAA 672 AUCUGCCAAGUCCUAAA AAGACATCAAATCT AGACAUCAAAUCU LDHA_exon4 − TTTG 96 TCTTCGATGACATCAA 673 UCUUCGAUGACAUCAAC CAAGAGCAAGTTCA AAGAGCAAGUUCA LDHA_exon4 − CTTC 97 GATGACATCAACAAGA 674 GAUGACAUCAACAAGAG GCAAGTTCATCTGC CAAGUUCAUCUGC LDHA_exon4 − CTTT 98 AGTTAAATGGAAAATT 675 AGUUAAAUGGAAAAUUG GCCACTTCTAGATT CCACUUCUAGAUU LDHA_exon4 − ATTT 99 GTCTTCGATGACATCA 676 GUCUUCGAUGACAUCAA ACAAGAGCAAGTTC CAAGAGCAAGUUC LDHA_exon4 − CTTT 100 GGTGTTCTAAGGAAAA 677 GGUGUUCUAAGGAAAAG GGCTGCCATGTTGG GCUGCCAUGUUGG LDHA_exon4 − TTTG 101 GTGTTCTAAGGAAAAG 678 GUGUUCUAAGGAAAAGG GCTGCCATGTTGGA CUGCCAUGUUGGA LDHA_exon4 − CTTT 102 GCCAGAGACAATCTTT 679 GCCAGAGACAAUCUUUG GGTGTTCTAAGGAA GUGUUCUAAGGAA LDHA_exon4 + ATTT 103 TCCATTTAACTAAAGA 680 UCCAUUUAACUAAAGAU TTTGATGTCTTTTA UUGAUGUCUUUUA LDHA_exon4 + TTTT 104 CCATTTAACTAAAGAT 681 CCAUUUAACUAAAGAUU TTGATGTCTTTTAG UGAUGUCUUUUAG LDHA_exon4 + TTTC 105 CATTTAACTAAAGATT 682 CAUUUAACUAAAGAUUU TGATGTCTTTTAGG GAUGUCUUUUAGG LDHA_exon4 + ATTT 106 AACTAAAGATTTGATG 683 AACUAAAGAUUUGAUGU TCTTTTAGGACTTG CUUUUAGGACUUG LDHA_exon4 + ATTT 107 GATGTCTTTTAGGACT 684 GAUGUCUUUUAGGACUU TGGCAGATGAACTT GGCAGAUGAACUU LDHA_exon4 + TTTG 108 ATGTCTTTTAGGACTT 685 AUGUCUUUUAGGACUUG GGCAGATGAACTTG GCAGAUGAACUUG LDHA_exon4 + CTTT 109 TAGGACTTGGCAGATG 686 UAGGACUUGGCAGAUGA AACTTGCTCTTGTT ACUUGCUCUUGUU LDHA_exon4 + TTTT 110 AGGACTTGGCAGATGA 687 AGGACUUGGCAGAUGAA ACTTGCTCTTGTTG CUUGCUCUUGUUG LDHA_exon4 + TTTA 111 GGACTTGGCAGATGAA 688 GGACUUGGCAGAUGAAC CTTGCTCTTGTTGA UUGCUCUUGUUGA LDHA_exon4 + CTTG 112 GCAGATGAACTTGCTC 689 GCAGAUGAACUUGCUCU TTGTTGATGTCATC UGUUGAUGUCAUC LDHA_exon4 + CTTG 113 CTCTTGTTGATGTCAT 690 CUCUUGUUGAUGUCAUC CGAAGACAAATTGA GAAGACAAAUUGA LDHA_exon4 + CTTG 114 TTGATGTCATCGAAGA 691 UUGAUGUCAUCGAAGAC CAAATTGAAGGGAG AAAUUGAAGGGAG LDHA_exon4 + GTTG 115 ATGTCATCGAAGACAA 692 AUGUCAUCGAAGACAAA ATTGAAGGGAGAGA UUGAAGGGAGAGA LDHA_exon4 + ATTG 116 AAGGGAGAGATGATGG 693 AAGGGAGAGAUGAUGGA ATCTCCAACATGGC UCUCCAACAUGGC LDHA_exon4 + CTTT 117 TCCTTAGAACACCAAA 694 UCCUUAGAACACCAAAG GATTGTCTCTGGCA AUUGUCUCUGGCA LDHA_exon4 + TTTT 118 CCTTAGAACACCAAAG 695 CCUUAGAACACCAAAGA ATTGTCTCTGGCAA UUGUCUCUGGCAA LDHA_exon4 + TTTC 119 CTTAGAACACCAAAGA 696 CUUAGAACACCAAAGAU TTGTCTCTGGCAAA UGUCUCUGGCAAA LDHA_exon4 + CTTA 120 GAACACCAAAGATTGT 697 GAACACCAAAGAUUGUC CTCTGGCAAAGGTT UCUGGCAAAGGUU LDHA_exon4 + ATTG 121 TCTCTGGCAAAGGTTG 698 UCUCUGGCAAAGGUUGA ATTTCAACAAGTTT UUUCAACAAGUUU LDHA_exon4 + GTTG 122 ATTTCAACAAGTTTAT 699 AUUUCAACAAGUUUAUA ATTATAATCCATGC UUAUAAUCCAUGC LDHA_exon4 + ATTT 123 CAACAAGTTTATATTA 700 CAACAAGUUUAUAUUAU TAATCCATGCTTGA AAUCCAUGCUUGA LDHA_exon4 + TTTC 124 AACAAGTTTATATTAT 701 AACAAGUUUAUAUUAUA AATCCATGCTTGAC AUCCAUGCUUGAC LDHA_exon4 + GTTT 125 ATATTATAATCCATGC 702 AUAUUAUAAUCCAUGCU TTGACTTAAATTCT UGACUUAAAUUCU LDHA_exon4 + TTTA 126 TATTATAATCCATGCT 703 UAUUAUAAUCCAUGCUU TGACTTAAATTCTT GACUUAAAUUCUU LDHA_exon4 − ATTT 127 AAGTCAAGCATGGATT 704 AAGUCAAGCAUGGAUUA ATAATATAAACTTG UAAUAUAAACUUG LDHA_exon4 − TTTA 128 AGTCAAGCATGGATTA 705 AGUCAAGCAUGGAUUAU TAATATAAACTTGT AAUAUAAACUUGU LDHA_exon4 − ATTA 129 TAATATAAACTTGTTG 706 UAAUAUAAACUUGUUGA AAATCAACCTTTGC AAUCAACCUUUGC LDHA_exon4 − CTTG 130 TTGAAATCAACCTTTG 707 UUGAAAUCAACCUUUGC CCAGAGACAATCTT CAGAGACAAUCUU LDHA_exon4 − GTTG 131 AAATCAACCTTTGCCA 708 AAAUCAACCUUUGCCAG GAGACAATCTTTGG AGACAAUCUUUGG LDHA_exon4 − TTTG 132 CCAGAGACAATCTTTG 709 CCAGAGACAAUCUUUGG GTGTTCTAAGGAAA UGUUCUAAGGAAA LDHA_exon4 + TTTA 133 ACTAAAGATTTGATGT 710 ACUAAAGAUUUGAUGUC CTTTTAGGACTTGG UUUUAGGACUUGG LDHA_exon4 + ATTA 134 TAATCCATGCTTGACT 711 UAAUCCAUGCUUGACUU TAAATTCTTT AAAUUCUUU LDHA_exon4 − TTTA 135 GTTAAATGGAAAATTG 712 GUUAAAUGGAAAAUUGC CCACTTCTAGATT CACUUCUAGAUU LDHA_exon4 − GTTA 136 AATGGAAAATTGCCAC 713 AAUGGAAAAUUGCCACU TTCTAGATT UCUAGAUU LDHA_exon5 + ATTT 137 ATTCTAAAGGCCTTAA 714 AUUCUAAAGGCCUUAAU TCTGGTCATTATTC CUGGUCAUUAUUC LDHA_exon5 − ATTA 138 TAGTCTAGAGAAAAGG 715 UAGUCUAGAGAAAAGGG GGAATAATGACCAG GAAUAAUGACCAG LDHA_exon5 + TTTT 139 GACTGCATAAAAATTG 716 GACUGCAUAAAAAUUGA ACAAGCTATAGTAA CAAGCUAUAGUAA LDHA_exon5 + GTTT 140 TGACTGCATAAAAATT 717 UGACUGCAUAAAAAUUG GACAAGGTATAGTA ACAAGCUAUAGUA LDHA_exon5 + TTTG 141 AAATCCAGGTGAGGCT 718 AAAUCCAGGUGAGGCUU TTTGACTGCATAAA UUGACUGCAUAAA LDHA_exon5 + GTTT 142 CAAATCCAGGTGAGGC 719 CAAAUCCAGGUGAGGCU TTTTGACTGCATAA UUUGACUGCAUAA LDHA_exon5 + ATTG 143 TTTCAAATCCAGGTGA 720 UUUCAAAUCCAGGUGAG GGCTTTTGACTGCA GCUUUUGACUGCA LDHA_exon5 + GTTA 144 TTGTTTCAAATCCAGG 721 UUGUUUCAAAUCCAGGU TGAGGCTTTTGACT GAGGCUUUUGACU LDHA_exon5 + GTTG 145 CTTATTGTTTCAAATC 722 CUUAUUGUUUCAAAUCC CAGGTGAGGCTTTT AGGUGAGGCUUUU LDHA_exon5 + GTTG 146 TAAAATACAGCCCGAA 723 UAAAAUACAGCCCGAAC CTGCAAGTTGCTTA UGCAAGUUGCUUA LDHA_exon5 + ATTC 147 CTAATGTTGTAAAATA 724 CUAAUGUUGUAAAAUAC CAGCCCGAACTGCA AGCCCGAACUGCA LDHA_exon5 + ATTC 148 ATCATTCCTAATGTTG 725 AUCAUUCCUAAUGUUGU TAAAATACAGCCCG AAAAUACAGCCCG LDHA_exon5 + TTTA 149 AATTCATCATTCCTAA 726 AAUUCAUCAUUCCUAAU TGTTGTAAAATACA GUUGUAAAAUACA LDHA_exon5 + TTTG 150 ACTGCATAAAAATTGA 727 ACUGCAUAAAAAUUGAC CAAGCTATAGTAAA AAGCUAUAGUAAA LDHA_exon5 + GTTT 151 AAATTCATCATTCCTA 728 AAAUUCAUCAUUCCUAA ATGTTGTAAAATAC UGUUGUAAAAUAC LDHA_exon5 + ATTT 152 GGTCCAGCGTAACGTG 729 GGUCCAGCGUAACGUGA AACATCTTTAAATT ACAUCUUUAAAUU LDHA_exon5 + CTTA 153 ATTTGGTCCAGCGTAA 730 AUUUGGUCCAGCGUAAC CGTGAACATCTTTA GUGAACAUCUUUA LDHA_exon5 + ATTA 154 TCACGGCTGGGGCACG 731 UCACGGCUGGGGCACGU TCAGCAAGAGGGAG CAGCAAGAGGGAG LDHA_exon5 + TTTC 155 TCTAGACTATAATGTA 732 UCUAGACUAUAAUGUAA ACTGCAAACTCCAA CUGCAAACUCCAA LDHA_exon5 + TTTT 156 CTCTAGACTATAATGT 733 CUCUAGACUAUAAUGUA AACTGCAAACTCCA ACUGCAAACUCCA LDHA_exon5 + CTTT 157 TCTCTAGACTATAATG 734 UCUCUAGACUAUAAUGU TAACTGCAAACTCC AACUGCAAACUCC LDHA_exon5 + ATTC 158 CCCTTTTCTCTAGACT 735 CCCUUUUCUCUAGACUA ATAATGTAACTGCA UAAUGUAACUGCA LDHA_exon5 + ATTA 159 TTCCCCTTTTCTCTAG 736 UUCCCCUUUUCUCUAGA ACTATAATGTAACT CUAUAAUGUAACU LDHA_exon5 + CTTA 160 ATCTGGTCATTATTCC 737 AUCUGGUCAUUAUUCCC CCTTTTCTCTAGAC CUUUUCUCUAGAC LDHA_exon5 + ATTC 161 TAAAGGCCTTAATCTG 738 UAAAGGCCUUAAUCUGG GTCATTATTCCCCT UCAUUAUUCCCCU LDHA_exon5 + TTTA 162 TTCTAAAGGCCTTAAT 739 UUCUAAAGGCCUUAAUC CTGGTCATTATTCC UGGUCAUUAUUCC LDHA_exon5 + TTTG 163 GTCCAGCGTAACGTGA 740 GUCCAGCGUAACGUGAA ACATCTTTAAATTC CAUCUUUAAAUUC LDHA_exon5 − TTTT 164 ACTATAGCTTGTCAAT 741 ACUAUAGCUUGUCAAUU TTTTATGCAGTCAA UUUAUGCAGUCAA LDHA_exon5 − GTTT 165 TACTATAGCTTGTCAA 742 UACUAUAGCUUGUCAAU TTTTTATGCAGTCA UUUUAUGCAGUCA LDHA_exon5 − ATTT 166 AAAGATGTTCACGTTA 743 AAAGAUGUUCACGUUAC CGCTGGACCAAATT GCUGGACCAAAUU LDHA_exon5 − GTTT 167 GCAGTTACATTATAGT 744 GCAGUUACAUUAUAGUC CTAGAGAAAAGGGG UAGAGAAAAGGGG LDHA_exon5 − CTTG 168 GAGTTTGCAGTTACAT 745 GAGUUUGCAGUUACAUU TATAGTCTAGAGAA AUAGUCUAGAGAA LDHA_exon5 − CTTG 169 CTGACGTGCCCCAGCC 746 CUGACGUGCCCCAGCCG GTGATAATGACCAG UGAUAAUGACCAG LDHA_exon5 − TTTC 170 TCCCTCTTGCTGACGT 747 UCCCUCUUGCUGACGUG GCCCCAGCCGTGAT CCCCAGCCGUGAU LDHA_exon5 − CTTT 171 CTCCCTCTTGCTGACG 748 CUCCCUCUUGCUGACGU TGCCCCAGCCGTGA GCCCCAGCCGUGA LDHA_exon5 − ATTA 172 AGACGGCTTTCTCCCT 749 AGACGGCUUUCUCCCUC CTTGCTGACGTGCC UUGCUGACGUGCC LDHA_exon5 − GTTA 173 CGCTGGACCAAATTAA 750 CGCUGGACCAAAUUAAG GACGGCTTTCTCCC ACGGCUUUCUCCC LDHA_exon5 − GTTC 174 ACGTTACGCTGGACCA 751 ACGUUACGCUGGACCAA AATTAAGACGGCTT AUUAAGACGGCUU LDHA_exon5 − TTTA 175 AAGATGTTCACGTTAC 752 AAGAUGUUCACGUUACG GCTGGACCAAATTA CUGGACCAAAUUA LDHA_exon5 − TTTA 176 CTATAGCTTGTCAATT 753 CUAUAGCUUGUCAAUUU TTTATGCAGTCAAA UUAUGCAGUCAAA LDHA_exon5 − ATTA 177 GGAATGATGAATTTAA 754 GGAAUGAUGAAUUUAAA AGATGTTCACGTTA GAUGUUCACGUUA LDHA_exon5 − TTTA 178 CAACATTAGGAATGAT 755 CAACAUUAGGAAUGAUG GAATTTAAAGATGT AAUUUAAAGAUGU LDHA_exon5 − TTTT 179 ACAACATTAGGAATGA 756 ACAACAUUAGGAAUGAU TGAATTTAAAGATG GAAUUUAAAGAUG LDHA_exon5 − ATTT 180 TACAACATTAGGAATG 757 UACAACAUUAGGAAUGA ATGAATTTAAAGAT UGAAUUUAAAGAU LDHA_exon5 − GTTC 181 GGGCTGTATTTTACAA 758 GGGCUGUAUUUUACAAC CATTAGGAATGATG AUUAGGAAUGAUG LDHA_exon5 − CTTG 182 CAGTTCGGGCTGTATT 759 CAGUUCGGGCUGUAUUU TTACAACATTAGGA UACAACAUUAGGA LDHA_exon5 − TTTG 183 AAACAATAAGCAACTT 760 AAACAAUAAGCAACUUG GCAGTTCGGGCTGT CAGUUCGGGCUGU LDHA_exon5 − ATTT 184 GAAACAATAAGCAACT 761 GAAACAAUAAGCAACUU TGCAGTTCGGGCTG GCAGUUCGGGCUG LDHA_exon5 − TTTA 185 TGCAGTCAAAAGCCTC 762 UGCAGUCAAAAGCCUCA ACCTGGATTTGAAA CCUGGAUUUGAAA LDHA_exon5 − TTTT 186 ATGCAGTCAAAAGCCT 763 AUGCAGUCAAAAGCCUC CACCTGGATTTGAA ACCUGGAUUUGAA LDHA_exon5 − TTTT 187 TATGCAGTCAAAAGCC 764 UAUGCAGUCAAAAGCCU TCACCTGGATTTGA CACCUGGAUUUGA LDHA_exon5 − ATTT 188 TTATGCAGTCAAAAGC 765 UUAUGCAGUCAAAAGCC CTCACCTGGATTTG UCACCUGGAUUUG LDHA_exon5 − CTTG 189 TCAATTTTTATGCAGT 766 UCAAUUUUUAUGCAGUC CAAAAGCCTCACCT AAAAGCCUCACCU LDHA_exon5 − TTTG 190 CAGTTACATTATAGTC 767 CAGUUACAUUAUAGUCU TAGAGAAAAGGGGA AGAGAAAAGGGGA LDHA_exon5 − GTTA 191 CATTATAGTCTAGAGA 768 CAUUAUAGUCUAGAGAA AAAGGGGAATAATG AAGGGGAAUAAUG LDHA_exon5 + ATTG 192 ACAAGCTATAGTAAAA 769 ACAAGCUAUAGUAAAAC CTGATAG UGAUAG LDHA_exon5 − ATTA 193 AGGCCTTTAGAATAAA 770 AGGCCUUUAGAAUAAAU TTTT UUU LDHA_exon6 − GTTA 194 TCTTCCAAGCCACGTA 771 UCUUCCAAGCCACGUAG GGTCAAGATATCCA GUCAAGAUAUCCA LDHA_exon6 − CTTG 195 CAAGCCACGTAGGTCA 772 CAAGCCACGUAGGUCAA AGATATCCACTATG GAUAUCCACUAUG LDHA_exon6 − TTTG 196 GGAAAACCACTTATCT 773 GGAAAACCACUUAUCUU TCCAAGCCACGTAG CCAAGCCACGUAG LDHA_exon6 + CTTG 197 ACCTACGTGGCTTGGA 774 ACCUACGUGGCUUGGAA AGATAAGTGGTTTT GAUAAGUGGUUUU LDHA_exon6 − TTTT 198 TGGGAAAACCACTTAT 775 UGGGAAAACCACUUAUC CTTCCAAGCCACGT UUCCAAGCCACGU LDHA_exon6 + GTTA 199 CCTAATGGGGGAAAGG 776 CCUAAUGGGGGAAAGGC CTGGGAGTTCACCC UGGGAGUUCACCC LDHA_exon6 + ATTC 200 CGTTACCTAATGGGGG 777 CGUUACCUAAUGGGGGA AAAGGCTGGGAGTT AAGGCUGGGAGUU LDHA_exon6 + ATTC 201 AGCCCGATTCCGTTAC 778 AGCCCGAUUCCGUUACC CTAATGGGGGAAAG UAAUGGGGGAAAG LDHA_exon6 + GTTG 202 CAATCTGGATTCAGCC 779 CAAUCUGGAUUCAGCCC CGATTCCGTTACCT GAUUCCGUUACCU LDHA_exon6 + ATTG 203 GAAGCGGTTGCAATCT 780 GAAGCGGUUGCAAUCUG GGATTCAGCCCGAT GAUUCAGCCCGAU LDHA_exon6 + TTTC 204 CCAAAAACCGTGTTAT 781 CCAAAAACCGUGUUAUU TGGAAGCGGTTGCA GGAAGCGGUUGCA LDHA_exon6 + TTTT 205 CCCAAAAACCGTGTTA 782 CCCAAAAACCGUGUUAU TTGGAAGCGGTTGC UGGAAGCGGUUGC LDHA_exon6 + GTTT 206 TCCCAAAAACCGTGTT 783 UCCCAAAAACCGUGUUA ATTGGAAGCGGTTG UUGGAAGCGGUUG LDHA_exon6 + CTTG 207 GAAGATAAGTGGTTTT 784 GAAGAUAAGUGGUUUUC CCCAAAAACCGTGT CCAAAAACCGUGU LDHA_exon6 + TTTC 208 ATAGTGGATATCTTGA 785 AUAGUGGAUAUCUUGAC CCTACGTGGCTTGG CUACGUGGCUUGG LDHA_exon6 + TTTT 209 CATAGTGGATATCTTG 786 CAUAGUGGAUAUCUUGA ACCTACGTGGCTTG CCUACGUGGCUUG LDHA_exon6 + TTTT 210 TCATAGTGGATATCTT 787 UCAUAGUGGAUAUCUUG GACCTACGTGGCTT ACCUACGUGGCUU LDHA_exon6 + GTTT 211 TTCATAGTGGATATCT 788 UUCAUAGUGGAUAUCUU TGACCTACGTGGCT GACCUACGUGGCU LDHA_exon6 + TTTC 212 TCCTTTTTCATAGTGG 789 UCCUUUUUCAUAGUGGA ATATCTTGACCTAC UAUCUUGACCUAC LDHA_exon6 + TTTT 213 CTCCTTTTTCATAGTG 790 CUCCUUUUUCAUAGUGG GATATCTTGACCTA AUAUCUUGACCUA LDHA_exon6 + ATTT 214 TCTCCTTTTTCATAGT 791 UCUCCUUUUUCAUAGUG GGATATCTTGACCT GAUAUCUUGACCU LDHA_exon6 + TTTA 215 TTTTCTCCTTTTTCAT 792 UUUUCUCCUUUUUCAUA AGTGGATATCTTGA GUGGAUAUCUUGA LDHA_exon6 + TTTT 216 ATTTTCTCCTTTTTCA 793 AUUUUCUCCUUUUUCAU TAGTGGATATCTTG AGUGGAUAUCUUG LDHA_exon6 + TTTT 217 TATTTTCTCCTTTTTC 794 UAUUUUCUCCUUUUUCA ATAGTGGATATCTT UAGUGGAUAUCUU LDHA_exon6 + ATTT 218 TTATTTTCTCCTTTTT 795 UUAUUUUCUCCUUUUUC CATAGTGGATATCT AUAGUGGAUAUCU LDHA_exon6 − TTTT 219 GGGAAAACCACTTATC 796 GGGAAAACCACUUAUCU TTCCAAGCCACGTA UCCAAGCCACGUA LDHA_exon6 + GTTC 220 ACCCATTAAGCTGTCA 797 ACCCAUUAAGCUGUCAU TGGGTGGGTCCTTG GGGUGGGUCCUUG LDHA_exon6 + ATTA 221 AGCTGTCATGGGTGGG 798 AGCUGUCAUGGGUGGGU TCCTTGGGGAACAT CCUUGGGGAACAU LDHA_exon6 + GTTA 222 TTGGAAGCGGTTGCAA 799 UUGGAAGCGGUUGCAAU TCTGGATTCAGCCC CUGGAUUCAGCCC LDHA_exon6 + ATTO 223 CAGTGGTAAGCATAAG 800 CAGUGGUAAGCAUAAGU TTATTTTCTTTTTG UAUUUUCUUUUUG LDHA_exon6 − GTTT 224 TTGGGAAAACCACTTA 801 UUGGGAAAACCACUUAU TCTTCCAAGCCACG CUUCCAAGCCACG LDHA_exon6 − CTTC 225 CAATAACACGGTTTTT 802 CAAUAACACGGUUUUUG GGGAAAACCACTTA GGAAAACCACUUA LDHA_exon6 − ATTG 226 CAACCGCTTCCAATAA 803 CAACCGCUUCCAAUAAC CACGGTTTTTGGGA ACGGUUUUUGGGA LDHA_exon6 − ATTA 227 GGTAACGGAATCGGGC 804 GGUAACGGAAUCGGGCU TGAATCCAGATTGC GAAUCCAGAUUGC LDHA_exon6 + CTTG 228 GGGAACATGGAGATTC 805 GGGAACAUGGAGAUUCC CAGTGGTAAGCATA AGUGGUAAGCAUA LDHA_exon6 − CTTT 229 CCCCCATTAGGTAACG 806 CCCCCAUUAGGUAACGG GAATCGGGCTGAAT AAUCGGGCUGAAU LDHA_exon6 − CTTA 230 ATGGGTGAACTCCCAG 807 AUGGGUGAACUCCCAGC CCTTTCCCCCATTA CUUUCCCCCAUUA LDHA_exon6 − GTTC 231 CCCAAGGACCCACCCA 808 CCCAAGGACCCACCCAU TGACAGCTTAATGG GACAGCUUAAUGG LDHA_exon6 − CTTA 232 CCACTGGAATCTCCAT 809 CCACUGGAAUCUCCAUG GTTCCCCAAGGACC UUCCCCAAGGACC LDHA_exon6 − CTTA 233 TGCTTACCACTGGAAT 810 UGCUUACCACUGGAAUC CTCCATGTTCCCCA UCCAUGUUCCCCA LDHA_exon6 − TTTC 234 AAAAACAAAAAGAAAA 811 AAAAACAAAAAGAAAAU TAACTTATGCTTAC AACUUAUGCUUAC LDHA_exon6 − TTTC 235 CCCCATTAGGTAACGG 812 CCCCAUUAGGUAACGGA AATCGGGCTGAATC AUCGGGCUGAAUC LDHA_exon6 + TTTT 236 CTTTTTGTTTTTGAAA 813 CUUUUUGUUUUUGAAAA AGATTATATAAAAA GAUUAUAUAAAAA LDHA_exon6 − CTTT 237 TCAAAAACAAAAAGAA 814 UCAAAAACAAAAAGAAA AATAACTTATGCTT AUAACUUAUGCUU LDHA_exon6 − TTTA 238 TATAATCTTTTCAAAA 815 UAUAAUCUUUUCAAAAA ACAAAAAGAAAATA CAAAAAGAAAAUA LDHA_exon6 − TTTT 239 ATATAATCTTTTCAAA 816 AUAUAAUCUUUUCAAAA AACAAAAAGAAAAT ACAAAAAGAAAAU LDHA_exon6 − TTTT 240 TATATAATCTTTTCAA 817 UAUAUAAUCUUUUCAAA AAACAAAAAGAAAA AACAAAAAGAAAA LDHA_exon6 − CTTT 241 TTATATAATCTTTTCA 818 UUAUAUAAUCUUUUCAA AAAACAAAAAGAAA AAACAAAAAGAAA LDHA_exon6 + TTTC 242 TTTTTGTTTTTGAAAA 819 UUUUUGUUUUUGAAAAG GATTATATAAAAAG AUUAUAUAAAAAG LDHA_exon6 + GTTA 243 TTTTCTTTTTGTTTTT 820 UUUUCUUUUUGUUUUUG GAAAAGATTATATA AAAAGAUUAUAUA LDHA_exon6 + ATTT 244 TCTTTTTGTTTTTGAA 821 UCUUUUUGUUUUUGAAA AAGATTATATAAAA AGAUUAUAUAAAA LDHA_exon6 − TTTT 245 CAAAAACAAAAAGAAA 822 CAAAAACAAAAAGAAAA ATAACTTATGCTTA UAACUUAUGCUUA LDHA_exon6 + TTTT 246 GAAAAGATTATATAAA 823 GAAAAGAUUAUAUAAAA AAGT AGU LDHA_exon6 + TTTT 247 TGAAAAGATTATATAA 824 UGAAAAGAUUAUAUAAA AAAGT AAGU LDHA_exon6 + GTTT 248 TTGAAAAGATTATATA 825 UUGAAAAGAUUAUAUAA AAAAGT AAAGU LDHA_exon6 + TTTT 249 GTTTTTGAAAAGATTA 826 GUUUUUGAAAAGAUUAU TATAAAAAGT AUAAAAAGU LDHA_exon6 + TTTT 250 TGTTTTTGAAAAGATT 827 UGUUUUUGAAAAGAUUA ATATAAAAAGT UAUAAAAAGU LDHA_exon6 + CTTT 251 TTGTTTTTGAAAAGAT 828 UUGUUUUUGAAAAGAUU TATATAAAAAGT AUAUAAAAAGU LDHA_exon6 + TTTG 252 TTTTTGAAAAGATTAT 829 UUUUUGAAAAGAUUAUA ATAAAAAGT UAAAAAGU LDHA_exon7 + GTTG 253 AGAGGTAATAAATCTT 830 AGAGGUAAUAAAUCUUU TCAATTTGGCAACA CAAUUUGGCAACA LDHA_exon7 + GTTG 254 GTACATGAAAATAAAT 831 GUACAUGAAAAUAAAUG GTAGTCTGTACTAT UAGUCUGUACUAU LDHA_exon7 + TTTC 255 AATTTGGCAACACAGA 832 AAUUUGGCAACACAGAA ATATTAACATTTAC UAUUAACAUUUAC LDHA_exon7 + GTTC 256 ACAAGCAGGTGGTTGA 833 ACAAGCAGGUGGUUGAG GAGGTAATAAATCT AGGUAAUAAAUCU LDHA_exon7 + ATTT 257 GGCAACACAGAATATT 834 GGCAACACAGAAUAUUA AACATTTACTATTT ACAUUUACUAUUU LDHA_exon7 + CTTT 258 CAATTTGGCAACACAG 835 CAAUUUGGCAACACAGA AATATTAACATTTA AUAUUAACAUUUA LDHA_exon7 + TTTA 259 GGGACTGATAAAGATA 836 GGGACUGAUAAAGAUAA AGGAACAGTGGAAA GGAACAGUGGAAA LDHA_exon7 + CTTT 260 TAGTGCCTGTATGGAG 837 UAGUGCCUGUAUGGAGU TGGAATGAATGTTG GGAAUGAAUGUUG LDHA_exon7 + GTTG 261 CTGGTGTCTCTCTGAA 838 CUGGUGUCUCUCUGAAG GACTCTGCACCCAG ACUCUGCACCCAG LDHA_exon7 + TTTA 262 GTGCCTGTATGGAGTG 839 GUGCCUGUAUGGAGUGG GAATGAATGTTGCT AAUGAAUGUUGCU LDHA_exon7 + TTTT 263 AGTGCCTGTATGGAGT 840 AGUGCCUGUAUGGAGUG GGAATGAATGTTGC GAAUGAAUGUUGC LDHA_exon7 + TTTC 264 TTTTAGTGCCTGTATG 841 UUUUAGUGCCUGUAUGG GAGTGGAATGAATG AGUGGAAUGAAUG LDHA_exon7 + ATTT 265 CTTTTAGTGCCTGTAT 842 CUUUUAGUGCCUGUAUG GGAGTGGAATGAAT GAGUGGAAUGAAU LDHA_exon7 + TTTG 266 GCAACACAGAATATTA 843 GCAACACAGAAUAUUAA ACATTTACTATTTT CAUUUACUAUUUU LDHA_exon7 + ATTT 267 AGGGACTGATAAAGAT 844 AGGGACUGAUAAAGAUA AAGGAACAGTGGAA AGGAACAGUGGAA LDHA_exon7 − GTTA 268 ATATTCTGTGTTGCCA 845 AUAUUCUGUGUUGCCAA AATTGAAAGATTTA AUUGAAAGAUUUA LDHA_exon7 − TTTA 269 TCAGTCCCTAAATCTG 846 UCAGUCCCUAAAUCUGG GGTGCAGAGTCTTC GUGCAGAGUCUUC LDHA_exon7 − GTTG 270 CCAAATTGAAAGATTT 847 CCAAAUUGAAAGAUUUA ATTACCTCTCAACC UUACCUCUCAACC LDHA_exon7 − ATTC 271 TGTGTTGCCAAATTGA 848 UGUGUUGCCAAAUUGAA AAGATTTATTACCT AGAUUUAUUACCU LDHA_exon7 − ATTC 272 CACTCCATACAGGCAC 849 CACUCCAUACAGGCACU TAAAAGAAATAGTA AAAAGAAAUAGUA LDHA_exon7 − OTTO 273 AGAGAGACACCAGCAA 850 AGAGAGACACCAGCAAC CATTCATTCCACTC AUUCAUUCCACUC LDHA_exon7 − CTTT 274 ATCAGTCCCTAAATCT 851 AUCAGUCCCUAAAUCUG GGGTGCAGAGTCTT GGUGCAGAGUCUU LDHA_exon7 − CTTA 275 TCTTTATCAGTCCCTA 852 UCUUUAUCAGUCCCUAA AATCTGGGTGCAGA AUCUGGGUGCAGA LDHA_exon7 − GTTC 276 CTTATCTTTATCAGTC 853 CUUAUCUUUAUCAGUCC CCTAAATCTGGGTG CUAAAUCUGGGUG LDHA_exon7 − ATTC 277 ATTCCACTCCATACAG 854 AUUCCACUCCAUACAGG GCACTAAAAGAAAT CACUAAAAGAAAU LDHA_exon7 − CTTT 278 CCACTGTTCCTTATCT 855 CCACUGUUCCUUAUCUU TTATCAGTCCCTAA UAUCAGUCCCUAA LDHA_exon7 − CTTG 279 TGAACCTCTTTCCACT 856 UGAACCUCUUUCCACUG GTTCCTTATCTTTA UUCCUUAUCUUUA LDHA_exon7 − ATTA 280 CCTCTCAACCACCTGC 857 CCUCUCAACCACCUGCU TTGTGAACCTCTTT UGUGAACCUCUUU LDHA_exon7 − TTTA 281 TTACCTCTCAACCACC 858 UUACCUCUCAACCACCU TGCTTGTGAACCTC GCUUGUGAACCUC LDHA_exon7 − ATTT 282 ATTACCTCTCAACCAC 859 AUUACCUCUCAACCACC CTGCTTGTGAACCT UGCUUGUGAACCU LDHA_exon7 − TTTC 283 CACTGTTCCTTATCTT 860 CACUGUUCCUUAUCUUU TATCAGTCCCTAAA AUCAGUCCCUAAA LDHA_exon7 − ATTG 284 AAAGATTTATTACCTC 861 AAAGAUUUAUUACCUCU TCAACCACCTGCTT CAACCACCUGCUU LDHA_exon7 − TTTA 285 TTTTCATGTACCAACA 862 UUUUCAUGUACCAACAG GATTAG AUUAG LDHA_exon7 − ATTT 286 ATTTTCATGTACCAAC 863 AUUUUCAUGUACCAACA AGATTAG GAUUAG LDHA_exon8 + ATTG 287 GACTCTCTGTAGCAGA 864 GACUCUCUGUAGCAGAU TTTGGCAGAGAGTA UUGGCAGAGAGUA LDHA_exon8 + CTTA 288 TGAGGTGATCAAACTC 865 UGAGGUGAUCAAACUCA AAAGGCTACACATC AAGGCUACACAUC LDHA_exon8 + TTTC 289 CTATCATACAGTGCTT 866 CUAUCAUACAGUGCUUA ATGAGGTGATCAAA UGAGGUGAUCAAA LDHA_exon8 + GTTT 290 CCTATCATACAGTGCT 867 CCUAUCAUACAGUGCUU TATGAGGTGATCAA AUGAGGUGAUCAA LDHA_exon8 + CTTT 291 ACCTATGGTTTCCTAT 868 ACCUAUGGUUUCCUAUC CATACAGTGCTTAT AUACAGUGCUUAU LDHA_exon8 + TTTC 292 TGCCTTTACCTATGGT 869 UGCCUUUACCUAUGGUU TTCCTATCATACAG UCCUAUCAUACAG LDHA_exon8 + TTTT 293 CTGCCTTTACCTATGG 870 CUGCCUUUACCUAUGGU TTTCCTATCATACA UUCCUAUCAUACA LDHA_exon8 + ATTT 294 GGCAGAGAGTATAATG 871 GGCAGAGAGUAUAAUGA AAGAATCTTAGGCG AGAAUCUUAGGCG LDHA_exon8 + TTTA 295 CCTATGGTTTCCTATC 872 CCUAUGGUUUCCUAUCA ATACAGTGCTTATG UACAGUGCUUAUG LDHA_exon8 + TTTG 296 GCAGAGAGTATAATGA 873 GCAGAGAGUAUAAUGAA AGAATCTTAGGCGG GAAUCUUAGGCGG LDHA_exon8 − CTTC 297 ATTATACTCTCTGCCA 874 AUUAUACUCUCUGCCAA AATCTGCTACAGAG AUCUGCUACAGAG LDHA_exon8 + GTTT 298 CCACCATGATTAAGGT 875 CCACCAUGAUUAAGGUA AGGTCTATGTAGTG GGUCUAUGUAGUG LDHA_exon8 + TTTC 299 CACCATGATTAAGGTA 876 CACCAUGAUUAAGGUAG GGTCTATGTAGTGA GUCUAUGUAGUGA LDHA_exon8 + ATTA 300 AGGTAGGTCTATGTAG 877 AGGUAGGUCUAUGUAGU TGATACGCTGCATT GAUACGCUGCAUU LDHA_exon8 − ATTC 301 AAATGCAGCGTATCAC 878 AAAUGCAGCGUAUCACU TACATAGACCTACC ACAUAGACCUACC LDHA_exon8 − CTTA 302 ATCATGGTGGAAACTG 879 AUCAUGGUGGAAACUGG GGTGCACCCGCCTA GUGCACCCGCCUA LDHA_exon8 − ATTC 303 TTCATTATACTCTCTG 880 UUCAUUAUACUCUCUGC CCAAATCTGCTACA CAAAUCUGCUACA LDHA_exon8 − ATTA 304 TACTCTCTGCCAAATC 881 UACUCUCUGCCAAAUCU TGCTACAGAGAGTC GCUACAGAGAGUC LDHA_exon8 − CTTT 305 GAGTTTGATCACCTCA 882 GAGUUUGAUCACCUCAU TAAGCACTGTATGA AAGCACUGUAUGA LDHA_exon8 − TTTG 306 AGTTTGATCACCTCAT 883 AGUUUGAUCACCUCAUA AAGCACTGTATGAT AGCACUGUAUGAU LDHA_exon8 + TTTT 307 TCTGCCTTTACCTATG 884 UCUGCCUUUACCUAUGG GTTTCCTATCATAC UUUCCUAUCAUAC LDHA_exon8 + CTTA 308 GGCGGGTGCACCCAGT 885 GGCGGGUGCACCCAGUU TTCCACCATGATTA UCCACCAUGAUUA LDHA_exon8 + CTTT 309 TTCTGCCTTTACCTAT 886 UUCUGCCUUUACCUAUG GGTTTCCTATCATA GUUUCCUAUCAUA LDHA_exon8 − TTTG 310 ATCACCTCATAAGCAC 887 AUCACCUCAUAAGCACU TGTATGATAGGAAA GUAUGAUAGGAAA LDHA_exon8 − GTTT 311 GATCACCTCATAAGCA 888 GAUCACCUCAUAAGCAC CTGTATGATAGGAA UGUAUGAUAGGAA LDHA_exon8 + ATTT 312 GAATGCTTTTTGCTGG 889 GAAUGCUUUUUGCUGGC CTTTT UUUU LDHA_exon8 + TTTG 313 AATGCTTTTTGCTGGC 890 AAUGCUUUUUGCUGGCU TTTT UUU LDHA_exon8 + CTTC 314 TGAGGAAGAGGCCCGT 891 UGAGGAAGAGGCCCGUU TTGAAGAAGAGTGC UGAAGAAGAGUGC LDHA_exon8 − TTTC 315 CAAATTAATATAATAA 892 CAAAUUAAUAUAAUAAC CTAGCAGCTTTATG UAGCAGCUUUAUG LDHA_exon9 − ATTA 316 ATATAATAACTAGCAG 893 AUAUAAUAACUAGCAGC CTTTATGACTTTAT UUUAUGACUUUAU LDHA_exon9 − CTTT 317 ATGACTTTATATCTTA 894 AUGACUUUAUAUCUUAA ATATAATGAATTAA UAUAAUGAAUUAA LDHA_exon9 − TTTA 318 TGACTTTATATCTTAA 895 UGACUUUAUAUCUUAAU TATAATGAATTAAC AUAAUGAAUUAAC LDHA_exon9 − CTTT 319 ATATCTTAATATAATG 896 AUAUCUUAAUAUAAUGA AATTAACCAAAGTA AUUAACCAAAGUA LDHA_exon9 − TTTA 320 TATCTTAATATAATGA 897 UAUCUUAAUAUAAUGAA ATTAACCAAAGTAG UUAACCAAAGUAG LDHA_exon9 − CTTA 321 ATATAATGAATTAACC 898 AUAUAAUGAAUUAACCA AAAGTAGTCACTGT AAGUAGUCACUGU LDHA_exon9 − ATTA 322 ACCAAAGTAGTCACTG 899 ACCAAAGUAGUCACUGU TTCAAGGTTTATTG UCAAGGUUUAUUG LDHA_exon9 − GTTC 323 AAGGTTTATTGGGGGT 900 AAGGUUUAUUGGGGGUU TTTAGTTGGTATAA UUAGUUGGUAUAA LDHA_exon9 − GTTT 324 ATTGGGGGTTTTAGTT 901 AUUGGGGGUUUUAGUUG GGTATAACACTTGG GUAUAACACUUGG LDHA_exon9 − TTTA 325 TTGGGGGTTTTAGTTG 902 UUGGGGGUUUUAGUUGG GTATAACACTTGGA UAUAACACUUGGA LDHA_exon9 − ATTG 326 GGGGTTTTAGTTGGTA 903 GGGGUUUUAGUUGGUAU TAACACTTGGATAG AACACUUGGAUAG LDHA_exon9 − GTTT 327 TAGTTGGTATAACACT 904 UAGUUGGUAUAACACUU TGGATAGTTGGTTG GGAUAGUUGGUUG LDHA_exon9 − ATTT 328 CCAAATTAATATAATA 905 CCAAAUUAAUAUAAUAA ACTAGCAGCTTTAT CUAGCAGCUUUAU LDHA_exon9 − TTTT 329 AGTTGGTATAACACTT 906 AGUUGGUAUAACACUUG GGATAGTTGGTTGC GAUAGUUGGUUGC LDHA_exon9 − GTTG 330 GTATAACACTTGGATA 907 GUAUAACACUUGGAUAG GTTGGTTGCATTGT UUGGUUGCAUUGU LDHA_exon9 − CTTG 331 GATAGTTGGTTGCATT 908 GAUAGUUGGUUGCAUUG GTTTGTATGTAGAT UUUGUAUGUAGAU LDHA_exon9 − GTTG 332 GTTGCATTGTTTGTAT 909 GUUGCAUUGUUUGUAUG GTAGATCTTTTTAC UAGAUCUUUUUAC LDHA_exon9 − GTTG 333 CATTGTTTGTATGTAG 910 CAUUGUUUGUAUGUAGA ATCTTTTTACATTA UCUUUUUACAUUA LDHA_exon9 − ATTG 334 TTTGTATGTAGATCTT 911 UUUGUAUGUAGAUCUUU TTTACATTATATGG UUACAUUAUAUGG LDHA_exon9 − GTTT 335 GTATGTAGATCTTTTT 912 GUAUGUAGAUCUUUUUA ACATTATATGGTAA CAUUAUAUGGUAA LDHA_exon9 − TTTG 336 TATGTAGATCTTTTTA 913 UAUGUAGAUCUUUUUAC CATTATATGGTAAT AUUAUAUGGUAAU LDHA_exon9 − CTTT 337 TTACATTATATGGTAA 914 UUACAUUAUAUGGUAAU TGTACACTACTGAT GUACACUACUGAU LDHA_exon9 − TTTT 338 TACATTATATGGTAAT 915 UACAUUAUAUGGUAAUG GTACACTACTGATA UACACUACUGAUA LDHA_exon9 − TTTT 339 ACATTATATGGTAATG 916 ACAUUAUAUGGUAAUGU TACACTACTGATAT ACACUACUGAUAU LDHA_exon9 − TTTA 340 CATTATATGGTAATGT CAUUAUAUGGUAAUGUA ACACTACTGATATA CACUACUGAUAUA LDHA_exon9 − ATTA 341 TATGGTAATGTACACT 918 UAUGGUAAUGUACACUA ACTGATATAGTTCA CUGAUAUAGUUCA LDHA_exon9 − GTTC 342 ACAAAATAAGATCCTT 919 ACAAAAUAAGAUCCUUU TGGAAGAATTATGC GGAAGAAUUAUGC LDHA_exon9 − CTTT 343 GGAAGAATTATGCACA 920 GGAAGAAUUAUGCACAA AGACATGATATTGG GACAUGAUAUUGG LDHA_exon9 − TTTA 344 GTTGGTATAACACTTG 921 GUUGGUAUAACACUUGG GATAGTTGGTTGCA AUAGUUGGUUGCA LDHA_exon9 − GTTG 345 CCCAAGAATAGCCTAA 922 CCCAAGAAUAGCCUAAU TATTTCCAAATTAA AUUUCCAAAUUAA LDHA_exon9 − GTTG 346 CAGGGTTGCCCAAGAA 923 CAGGGUUGCCCAAGAAU TAGCCTAATATTTC AGCCUAAUAUUUC LDHA_exon9 − GTTA 347 GAAAAAATCGTTGCAG 924 GAAAAAAUCGUUGCAGG GGTTGCCCAAGAAT GUUGCCCAAGAAU LDHA_exon9 − ATTG 348 TTTTTAATTGTTACCA 925 UUUUUAAUUGUUACCAG GCTTCCAGAGGACA CUUCCAGAGGACA LDHA_exon9 − GTTT 349 TTAATTGTTACCAGCT 926 UUAAUUGUUACCAGCUU TCCAGAGGACAAGA CCAGAGGACAAGA LDHA_exon9 − TTTT 350 TAATTGTTACCAGCTT 927 UAAUUGUUACCAGCUUC CCAGAGGACAAGAT CAGAGGACAAGAU LDHA_exon9 − TTTT 351 AATTGTTACCAGCTTC 928 AAUUGUUACCAGCUUCC CAGAGGACAAGATC AGAGGACAAGAUC LDHA_exon9 − TTTA 352 ATTGTTACCAGCTTCC 929 AUUGUUACCAGCUUCCA AGAGGACAAGATCT GAGGACAAGAUCU LDHA_exon9 − ATTG 353 TTACCAGCTTCCAGAG 930 UUACCAGCUUCCAGAGG GACAAGATCTCAAA ACAAGAUCUCAAA LDHA_exon9 − GTTA 354 CCAGCTTCCAGAGGAC 931 CCAGCUUCCAGAGGACA AAGATCTCAAAAAT AGAUCUCAAAAAU LDHA_exon9 − GTTG 355 CAGAGGACAAGATCTC 932 CAGAGGACAAGAUCUCA AAAAATCTGTGTTC AAAAUCUGUGUUC LDHA_exon9 − GTTG 356 CCTATAGTGACACACT 933 CCUAUAGUGACACACUA ATCATTGCCTATAT UCAUUGCCUAUAU LDHA_exon9 − ATTG 357 CCTATATTCAGTTGGC 934 CCUAUAUUCAGUUGGCA AAATAAATTTTACA AAUAAAUUUUACA LDHA_exon9 − ATTG 358 AGTTGGCAAATAAATT 935 AGUUGGCAAAUAAAUUU TTACATTTACATAT UACAUUUACAUAU LDHA_exon9 − GTTG 359 GCAAATAAATTTTACA 936 GCAAAUAAAUUUUACAU TTTACATATAGAAT UUACAUAUAGAAU LDHA_exon9 − ATTT 360 TACATTTACATATAGA 937 UACAUUUACAUAUAGAA ATGTTACTTTCCAA UGUUACUUUCCAA LDHA_exon9 − TTTT 361 ACATTTACATATAGAA 938 ACAUUUACAUAUAGAAU TGTTACTTTCCAAT GUUACUUUCCAAU LDHA_exon9 − TTTG 362 GAAGAATTATGCACAA 939 GAAGAAUUAUGCACAAG GACATGATATTGGA ACAUGAUAUUGGA LDHA_exon9 − TTTA 363 CATTTACATATAGAAT 940 CAUUUACAUAUAGAAUG GTTACTTTCCAATT UUACUUUCCAAUU LDHA_exon9 − TTTA 364 CATATAGAATGTTACT 941 CAUAUAGAAUGUUACUU TTCCAATTATGATT UCCAAUUAUGAUU LDHA_exon9 − GTTA 365 CTTTCCAATTATGATT 942 CUUUCCAAUUAUGAUUA AGCATTATTATCAA GCAUUAUUAUCAA LDHA_exon9 − CTTT 366 CCAATTATGATTAGCA 943 CCAAUUAUGAUUAGCAU TTATTATCAAATAT UAUUAUCAAAUAU LDHA_exon9 − TTTC 367 CAATTATGATTAGCAT 944 CAAUUAUGAUUAGCAUU TATTATCAAATATA AUUAUCAAAUAUA LDHA_exon9 − ATTA 368 TGATTAGCATTATTAT 945 UGAUUAGCAUUAUUAUC CAAATATATAATAC AAAUAUAUAAUAC LDHA_exon9 − ATTA 369 GCATTATTATCAAATA 946 GCAUUAUUAUCAAAUAU TATAATACTTTGGG AUAAUACUUUGGG LDHA_exon9 − ATTA 370 TTATCAAATATATAAT 947 UUAUCAAAUAUAUAAUA ACTTTGGGACTTAC CUUUGGGACUUAC LDHA_exon9 − ATTA 371 TCAAATATATAATACT 948 UCAAAUAUAUAAUACUU TTGGGACTTACAAT UGGGACUUACAAU LDHA_exon9 − CTTT 372 GGGACTTACAATGGAA 949 GGGACUUACAAUGGAAG GTGGTACCAATACA UGGUACCAAUACA LDHA_exon9 − TTTG 373 GGACTTACAATGGAAG 950 GGACUUACAAUGGAAGU TGGTACCAATACAA GGUACCAAUACAA LDHA_exon9 − CTTA 374 CAATGGAAGTGGTACC 951 CAAUGGAAGUGGUACCA AATACAACTCAGTT AUACAACUCAGUU LDHA_exon9 − GTTG 375 ACTATTACATCCTCTG 952 ACUAUUACAUCCUCUGC CTATTAGTCAATAA UAUUAGUCAAUAA LDHA_exon9 − ATTA 376 CATCCTCTGCTATTAG 953 CAUCCUCUGCUAUUAGU TCAATAATATCCCT CAAUAAUAUCCCU LDHA_exon9 − ATTA 377 GTCAATAATATCCCTG 954 GUCAAUAAUAUCCCUGU TTAGAAAAAATCGT UAGAAAAAAUCGU LDHA_exon9 − ATTT 378 ACATATAGAATGTTAC 955 ACAUAUAGAAUGUUACU TTTCCAATTATGAT UUCCAAUUAUGAU LDHA_exon9 − ATTA 379 TGCACAAGACATGATA 956 UGCACAAGACAUGAUAU TTGGATTTATACAC UGGAUUUAUACAC LDHA_exon9 − ATTG 380 GATTTATACACTGGAT 957 GAUUUAUACACUGGAUC CCCAGGATGTGACT CCAGGAUGUGACU LDHA_exon9 − ATTT 381 ATACACTGGATCCCAG 958 AUACACUGGAUCCCAGG GATGTGACTCACTG AUGUGACUCACUG LDHA_exon9 − CTTC 382 AAACGGGCCTCTTCCT 959 AAACGGGCCUCUUCCUC CAGAAGTCAGAGTC AGAAGUCAGAGUC LDHA_exon9 − CTTC 383 CTCAGAAGTCAGAGTC 960 CUCAGAAGUCAGAGUCA ACCTTCACAAGGTC CCUUCACAAGGUC LDHA_exon9 − CTTC 384 ACAAGGTCTGAGATTC 961 ACAAGGUCUGAGAUUCC CATTCTGTCCCAAA AUUCUGUCCCAAA LDHA_exon9 − ATTC 385 CATTCTGTCCCAAAAT 962 CAUUCUGUCCCAAAAUG GCAAGGAACACTAA CAAGGAACACUAA LDHA_exon9 − ATTC 386 TGTCCCAAAATGCAAG 963 UGUCCCAAAAUGCAAGG GAACACTAAGGAAG AACACUAAGGAAG LDHA_exon9 − CTTT 387 ATTCCGTAAAGACCCT 964 AUUCCGUAAAGACCCUG GAAGATGAAATGAA AAGAUGAAAUGAA LDHA_exon9 − TTTA 388 TTCCGTAAAGACCCTG 965 UUCCGUAAAGACCCUGA AAGATGAAATGAAA AGAUGAAAUGAAA LDHA_exon9 − ATTC 389 CGTAAAGACCCTGAAG 966 CGUAAAGACCCUGAAGA ATGAAATGAAAAAA UGAAAUGAAAAAA LDHA_exon9 + TTTG 390 GGACAGAATGGAATCT 967 GGACAGAAUGGAAUCUC CAGACCTTGTGAAG AGACCUUGUGAAG LDHA_exon9 + TTTT 391 GGGACAGAATGGAATC 968 GGGACAGAAUGGAAUCU TCAGACCTTGTGAA CAGACCUUGUGAA LDHA_exon9 + ATTT 392 TGGGACAGAATGGAAT 969 UGGGACAGAAUGGAAUC CTCAGACCTTGTGA UCAGACCUUGUGA LDHA_exon9 + CTTG 393 CATTTTGGGACAGAAT 970 CAUUUUGGGACAGAAUG GGAATCTCAGACCT GAAUCUCAGACCU LDHA_exon9 + GTTC 394 CTTGCATTTTGGGACA 971 CUUGCAUUUUGGGACAG GAATGGAATCTCAG AAUGGAAUCUCAG LDHA_exon9 + CTTC 395 CTTAGTGTTCCTTGCA 972 CUUAGUGUUCCUUGCAU TTTTGGGACAGAAT UUUGGGACAGAAU LDHA_exon9 − CTTC 396 TTCAAACGGGCCTCTT 973 UUCAAACGGGCCUCUUC CCTCAGAAGTCAGA CUCAGAAGUCAGA LDHA_exon9 + TTTA 397 CGGAATAAAGGATGAT 974 CGGAAUAAAGGAUGAUG GTCTTCCTTAGTGT UCUUCCUUAGUGU LDHA_exon9 + CTTC 398 AGGGTCTTTACGGAAT 975 AGGGUCUUUACGGAAUA AAAGGATGATGTCT AAGGAUGAUGUCU LDHA_exon9 + TTTC 399 ATCTTCAGGGTCTTTA 976 AUCUUCAGGGUCUUUAC CGGAATAAAGGATG GGAAUAAAGGAUG LDHA_exon9 + ATTT 400 CATCTTCAGGGTCTTT 977 CAUCUUCAGGGUCUUUA ACGGAATAAAGGAT CGGAAUAAAGGAU LDHA_exon9 + TTTC 401 ATTTCATCTTCAGGGT 978 AUUUCAUCUUCAGGGUC CTTTACGGAATAAA UUUACGGAAUAAA LDHA_exon9 + TTTT 402 CATTTCATCTTCAGGG 979 CAUUUCAUCUUCAGGGU TCTTTACGGAATAA CUUUACGGAAUAA LDHA_exon9 + TTTT 403 TCATTTCATCTTCAGG 980 UCAUUUCAUCUUCAGGG GTCTTTACGGAATA UCUUUACGGAAUA LDHA_exon9 + TTTT 404 TTCATTTCATCTTCAG 981 UUCAUUUCAUCUUCAGG GGTCTTTACGGAAT GUCUUUACGGAAU LDHA_exon9 + TTTT 405 TTTCATTTCATCTTCA 982 UUUCAUUUCAUCUUCAG GGGTCTTTACGGAA GGUCUUUACGGAA LDHA_exon9 + TTTT 406 TTTTCATTTCATCTTC 983 UUUUCAUUUCAUCUUCA AGGGTCTTTACGGA GGGUCUUUACGGA LDHA_exon9 + TTTT 407 TTTTTCATTTCATCTT 984 UUUUUCAUUUCAUCUUC CAGGGTCTTTACGG AGGGUCUUUACGG LDHA_exon9 + TTTT 408 TTTTTTCATTTCATCT 985 UUUUUUCAUUUCAUCUU TCAGGGTCTTTACG CAGGGUCUUUACG LDHA_exon9 + TTTT 409 TTTTTTTCATTTCATC 986 UUUUUUUCAUUUCAUCU TTCAGGGTCTTTAC UCAGGGUCUUUAC LDHA_exon9 + TTTT 410 TTTTTTTTCATTTCAT 987 UUUUUUUUCAUUUCAUC CTTCAGGGTCTTTA UUCAGGGUCUUUA LDHA_exon9 + ATTT 411 TTTTTTTTTCATTTCA 988 UUUUUUUUUCAUUUCAU TCTTCAGGGTCTTT CUUCAGGGUCUUU LDHA_exon9 + CTTT 412 ACGGAATAAAGGATGA 989 ACGGAAUAAAGGAUGAU TGTCTTCCTTAGTG GUCUUCCUUAGUG LDHA_exon9 − CTTA 413 AGATTGTTTTTAATTG 990 AGAUUGUUUUUAAUUGU TTACCAGCTTCCAG UACCAGCUUCCAG LDHA_exon9 − TTTG 414 GATCCCCCAAAGTGTA 991 GAUCCCCCAAAGUGUAU TCTGCACTCTTCTT CUGCACUCUUCUU LDHA_exon9 − CTTT 415 TGGATCCCCCAAAGTG 992 UGGAUCCCCCAAAGUGU TATCTGCACTCTTC AUCUGCACUCUUC LDHA_exon9 − TTTA 416 TACACTGGATCCCAGG 993 UACACUGGAUCCCAGGA ATGTGACTCACTGG UGUGACUCACUGG LDHA_exon9 − GTTG 417 GACTAGGCATGTTCAG 994 GACUAGGCAUGUUCAGU TGAAGGAGCCAGGA GAAGGAGCCAGGA LDHA_exon9 − GTTC 418 AGTGAAGGAGCCAGGA 995 AGUGAAGGAGCCAGGAA AGTTATATAACACA GUUAUAUAACACA LDHA_exon9 − GTTA 419 TATAACACACGGTAAA 996 UAUAACACACGGUAAAC CATCCACCTGGCTC AUCCACCUGGCUC LDHA_exon9 − ATTG 420 GCAGTGGTGCGTCAGA 997 GCAGUGGUGCGUCAGAG GGTGGCAGAACTAT GUGGCAGAACUAU LDHA_exon9 − ATTT 421 CACACTAACCAGTTGA 998 CACACUAACCAGUUGAA AGACTACACAAGAT GACUACACAAGAU LDHA_exon9 − TTTC 422 ACACTAACCAGTTGAA 999 ACACUAACCAGUUGAAG GACTACACAAGATT ACUACACAAGAUU LDHA_exon9 − GTTG 423 AAGACTACACAAGATT 1000 AAGACUACACAAGAUUA AATACCATCCAGCA AUACCAUCCAGCA LDHA_exon9 − ATTA 424 ATACCATCCAGCATCA 1001 AUACCAUCCAGCAUCAG GGATATAGCTGTGG GAUAUAGCUGUGG LDHA_exon9 − ATTT 425 TACAAACCATTCTTAT 1002 UACAAACCAUUCUUAUU TTCTAACTTCAGGA UCUAACUUCAGGA LDHA_exon9 − TTTT 426 ACAAACCATTCTTATT 1003 ACAAACCAUUCUUAUUU TCTAACTTCAGGAG CUAACUUCAGGAG LDHA_exon9 − TTTA 427 CAAACCATTCTTATTT 1004 CAAACCAUUCUUAUUUC CTAACTTCAGGAGT UAACUUCAGGAGU LDHA_exon9 − ATTC 428 TTATTTCTAACTTCAG 1005 UUAUUUCUAACUUCAGG GAGTTGATGTTTTT AGUUGAUGUUUUU LDHA_exon9 − CTTA 429 TTTCTAACTTCAGGAG 1006 UUUCUAACUUCAGGAGU TTGATGTTTTTCCC UGAUGUUUUUCCC LDHA_exon9 − TTTT 430 GGATCCCCCAAAGTGT 1007 GGAUCCCCCAAAGUGUA ATCTGCACTCTTCT UCUGCACUCUUCU LDHA_exon9 − ATTT 431 CTAACTTCAGGAGTTG 1008 CUAACUUCAGGAGUUGA ATGTTTTTCCCAGT UGUUUUUCCCAGU LDHA_exon9 − CTTC 432 AGGAGTTGATGTTTTT 1009 AGGAGUUGAUGUUUUUC CCCAGTCCATCTTA CCAGUCCAUCUUA LDHA_exon9 − GTTG 433 ATGTTTTTCCCAGTCC 1010 AUGUUUUUCCCAGUCCA ATCTTAAAATATTA UCUUAAAAUAUUA LDHA_exon9 − GTTT 434 TTCCCAGTCCATCTTA 1011 UUCCCAGUCCAUCUUAA AAATATTACTGCTT AAUAUUACUGCUU LDHA_exon9 − TTTT 435 TCCCAGTCCATCTTAA 1012 UCCCAGUCCAUCUUAAA AATATTACTGCTTT AUAUUACUGCUUU LDHA_exon9 − TTTT 436 CCCAGTCCATCTTAAA 1013 CCCAGUCCAUCUUAAAA ATATTACTGCTTTA UAUUACUGCUUUA LDHA_exon9 − TTTC 437 CCAGTCCATCTTAAAA 1014 CCAGUCCAUCUUAAAAU TATTACTGCTTTAA AUUACUGCUUUAA LDHA_exon9 − CTTA 438 AAATATTACTGCTTTA 1015 AAAUAUUACUGCUUUAA ATCACAGATCAGAT UCACAGAUCAGAU LDHA_exon9 − ATTA 439 CTGCTTTAATCACAGA 1016 CUGCUUUAAUCACAGAU TCAGATAAAAAGGA CAGAUAAAAAGGA LDHA_exon9 − CTTT 440 AATCACAGATCAGATA 1017 AAUCACAGAUCAGAUAA AAAAGGACAACATG AAAGGACAACAUG LDHA_exon9 − TTTA 441 ATCACAGATCAGATAA 1018 AUCACAGAUCAGAUAAA AAAGGACAACATGC AAGGACAACAUGC LDHA_exon9 − GTTG 442 TAGCCTAGACAGTGAA 1019 UAGCCUAGACAGUGAAA ATGATATGACATCA UGAUAUGACAUCA LDHA_exon9 − CTTT 443 AAAATTGCAGCTCCTT 1020 AAAAUUGCAGCUCCUUU TTGGATCCCCCAAA UGGAUCCCCCAAA LDHA_exon9 − TTTA 444 AAATTGCAGCTCCTTT 1021 AAAUUGCAGCUCCUUUU TGGATCCCCCAAAG GGAUCCCCCAAAG LDHA_exon9 − ATTG 445 CAGCTCCTTTTGGATC 1022 CAGCUCCUUUUGGAUCC CCCCAAAGTGTATC CCCAAAGUGUAUC LDHA_exon9 − TTTC 446 TAACTTCAGGAGTTGA 1023 UAACUUCAGGAGUUGAU TGTTTTTCCCAGTC GUUUUUCCCAGUC LDHA_exon9 + CTTG 447 TGAAGGTGACTCTGAC 1024 UGAAGGUGACUCUGACU TTCTGAGGAAGAGG UCUGAGGAAGAGG LDHA_exon9 − ATTA 448 TAGGCATGAGCCACTG 1025 UAGGCAUGAGCCACUGC CACCCTGCCTTAAG ACCCUGCCUUAAG LDHA_exon9 − ATTC 449 CTGGCCTCCAGTGATC 1026 CUGGCCUCCAGUGAUCA AGCCCACCTGGGCT GCCCACCUGGGCU LDHA_exon9 + GTTA 450 TATAACTTCCTGGCTC 1027 UAUAACUUCCUGGCUCC CTTCACTGAACATG UUCACUGAACAUG LDHA_exon9 + CTTC 451 CTGGCTCCTTCACTGA 1028 CUGGCUCCUUCACUGAA ACATGCCTAGTCCA CAUGCCUAGUCCA LDHA_exon9 + CTTC 452 ACTGAACATGCCTAGT 1029 ACUGAACAUGCCUAGUC CCAACATTTTTTCC CAACAUUUUUUCC LDHA_exon9 + ATTT 453 TTTCCCAGTGAGTCAC 1030 UUUCCCAGUGAGUCACA ATCCTGGGATCCAG UCCUGGGAUCCAG LDHA_exon9 + TTTT 454 TTCCCAGTGAGTCACA 1031 UUCCCAGUGAGUCACAU TCCTGGGATCCAGT CCUGGGAUCCAGU LDHA_exon9 + TTTT 455 TCCCAGTGAGTCACAT 1032 UCCCAGUGAGUCACAUC CCTGGGATCCAGTG CUGGGAUCCAGUG LDHA_exon9 + TTTT 456 CCCAGTGAGTCACATC 1033 CCCAGUGAGUCACAUCC CTGGGATCCAGTGT UGGGAUCCAGUGU LDHA_exon9 + TTTC 457 CCAGTGAGTCACATCC 1034 CCAGUGAGUCACAUCCU TGGGATCCAGTGTA GGGAUCCAGUGUA LDHA_exon9 + CTTG 458 TGCATAATTCTTCCAA 1035 UGCAUAAUUCUUCCAAA AGGATCTTATTTTG GGAUCUUAUUUUG LDHA_exon9 + ATTC 459 TTCCAAAGGATCTTAT 1036 UUCCAAAGGAUCUUAUU TTTGTGAACTATAT UUGUGAACUAUAU LDHA_exon9 + CTTC 460 CAAAGGATCTTATTTT 1037 CAAAGGAUCUUAUUUUG GTGAACTATATCAG UGAACUAUAUCAG LDHA_exon9 + CTTA 461 TTTTGTGAACTATATC 1038 UUUUGUGAACUAUAUCA AGTAGTGTACATTA GUAGUGUACAUUA LDHA_exon9 + ATTT 462 TGTGAACTATATCAGT 1039 UGUGAACUAUAUCAGUA AGTGTACATTACCA GUGUACAUUACCA LDHA_exon9 + TTTT 463 GTGAACTATATCAGTA 1040 GUGAACUAUAUCAGUAG GTGTACATTACCAT UGUACAUUACCAU LDHA_exon9 + TTTA 464 CCGTGTGTTATATAAC 1041 CCGUGUGUUAUAUAACU TTCCTGGCTCCTTC UCCUGGCUCCUUC LDHA_exon9 + TTTG 465 TGAACTATATCAGTAG 1042 UGAACUAUAUCAGUAGU TGTACATTACCATA GUACAUUACCAUA LDHA_exon9 + GTTA 466 TACCAACTAAAACCCC 1043 UACCAACUAAAACCCCC CAATAAACCTTGAA AAUAAACCUUGAA LDHA_exon9 + CTTG 467 AACAGTGACTACTTTG 1044 AACAGUGACUACUUUGG GTTAATTCATTATA UUAAUUCAUUAUA LDHA_exon9 + CTTT 468 GGTTAATTCATTATAT 1045 GGUUAAUUCAUUAUAUU TAAGATATAAAGTC AAGAUAUAAAGUC LDHA_exon9 + TTTG 469 GTTAATTCATTATATT 1046 GUUAAUUCAUUAUAUUA AAGATATAAAGTCA AGAUAUAAAGUCA LDHA_exon9 + GTTA 470 ATTCATTATATTAAGA 1047 AUUCAUUAUAUUAAGAU TATAAAGTCATAAA AUAAAGUCAUAAA LDHA_exon9 + ATTC 471 ATTATATTAAGATATA 1048 AUUAUAUUAAGAUAUAA AAGTCATAAAGCTG AGUCAUAAAGCUG LDHA_exon9 + ATTA 472 TATTAAGATATAAAGT 1049 UAUUAAGAUAUAAAGUC CATAAAGCTGCTAG AUAAAGCUGCUAG LDHA_exon9 + ATTA 473 AGATATAAAGTCATAA 1050 AGAUAUAAAGUCAUAAA AGCTGCTAGTTATT GCUGCUAGUUAUU LDHA_exon9 + GTTA 474 TTATATTAATTTGGAA 1051 UUAUAUUAAUUUGGAAA ATATTAGGCTATTC UAUUAGGCUAUUC LDHA_exon9 + ATTA 475 TATTAATTTGGAAATA 1052 UAUUAAUUUGGAAAUAU TTAGGCTATTCTTG UAGGCUAUUCUUG LDHA_exon9 + ATTA 476 ATTTGGAAATATTAGG 1053 AUUUGGAAAUAUUAGGC CTATTCTTGGGCAA UAUUCUUGGGCAA LDHA_exon9 + ATTT 477 GGAAATATTAGGCTAT 1054 GGAAAUAUUAGGCUAUU TCTTGGGCAACCCT CUUGGGCAACCCU LDHA_exon9 + TTTG 478 GAAATATTAGGCTATT 1055 GAAAUAUUAGGCUAUUC CTTGGGCAACCCTG UUGGGCAACCCUG LDHA_exon9 + ATTA 479 GGCTATTCTTGGGCAA 1056 GGCUAUUCUUGGGCAAC CCCTGCAACGATTT CCUGCAACGAUUU LDHA_exon9 + ATTA 480 CCATATAATGTAAAAA 1057 CCAUAUAAUGUAAAAAG GATCTACATACAAA AUCUACAUACAAA LDHA_exon9 + ATTC 481 TTGGGCAACCCTGCAA 1058 UUGGGCAACCCUGCAAC CGATTTTTTCTAAC GAUUUUUUCUAAC LDHA_exon9 + GTTT 482 ACCGTGTGTTATATAA 1059 ACCGUGUGUUAUAUAAC CTTCCTGGCTCCTT UUCCUGGCUCCUU LDHA_exon9 + TTTG 483 CCCCTTGAGCCAGGTG 1060 CCCCUUGAGCCAGGUGG GATGTTTACCGTGT AUGUUUACCGUGU LDHA_exon9 + GTTT 484 GAAGAAGAGTGCAGAT 1061 GAAGAAGAGUGCAGAUA ACACTTTGGGGGAT CACUUUGGGGGAU LDHA_exon9 + TTTG 485 AAGAAGAGTGCAGATA 1062 AAGAAGAGUGCAGAUAC CACTTTGGGGGATC ACUUUGGGGGAUC LDHA_exon9 + GTTT 486 GGGGGATCCAAAAGGA 1063 GGGGGAUCCAAAAGGAG GCTGCAATTTTAAA CUGCAAUUUUAAA LDHA_exon9 + TTTG 487 GGGGATCCAAAAGGAG 1064 GGGGAUCCAAAAGGAGC CTGCAATTTTAAAG UGCAAUUUUAAAG LDHA_exon9 + ATTT 488 TAAAGTCTTCTGATGT 1065 UAAAGUCUUCUGAUGUC CATATCATTTCACT AUAUCAUUUCACU LDHA_exon9 + TTTT 489 AAAGTCTTCTGATGTC 1066 AAAGUCUUCUGAUGUCA ATATCATTTCAGTG UAUCAUUUCACUG LDHA_exon9 + TTTA 490 AAGTCTTCTGATGTCA 1067 AAGUCUUCUGAUGUCAU TATCATTTCACTGT AUCAUUUCACUGU LDHA_exon9 + CTTC 491 TGATGTCATATCATTT 1068 UGAUGUCAUAUCAUUUC CACTGTCTAGGCTA ACUGUCUAGGCUA LDHA_exon9 + ATTT 492 CACTGTCTAGGCTACA 1069 CACUGUCUAGGCUACAA ACAGGATTCTAGGT CAGGAUUCUAGGU LDHA_exon9 + TTTG 493 ACTGTCTAGGCTACAA 1070 ACUGUCUAGGCUACAAC CAGGATTCTAGGTG AGGAUUCUAGGUG LDHA_exon9 + ATTC 494 TAGGTGGAGGTTGTGC 1071 UAGGUGGAGGUUGUGCA ATGTTGTCCTTTTT UGUUGUCCUUUUU LDHA_exon9 + GTTG 495 TGCATGTTGTCCTTTT 1072 UGCAUGUUGUCCUUUUU TATCTGATCTGTGA AUCUGAUCUGUGA LDHA_exon9 + GTTG 496 TCCTTTTTATCTGATC 1073 UCCUUUUUAUCUGAUCU TGTGATTAAAGCAG GUGAUUAAAGCAG LDHA_exon9 + GTTT 497 TTATCTGATCTGTGAT 1074 UUAUCUGAUCUGUGAUU TAAAGCAGTAATAT AAAGCAGUAAUAU LDHA_exon9 + GTTG 498 AGCCAGGTGGATGTTT 1075 AGCCAGGUGGAUGUUUA ACCGTGTGTTATAT CCGUGUGUUAUAU LDHA_exon9 + TTTT 499 TATCTGATCTGTGATT 1076 UAUCUGAUCUGUGAUUA AAAGCAGTAATATT AAGCAGUAAUAUU LDHA_exon9 + TTTA 500 TCTGATCTGTGATTAA 1077 UCUGAUCUGUGAUUAAA AGCAGTAATATTTT GCAGUAAUAUUUU LDHA_exon9 + ATTA 501 AAGCAGTAATATTTTA 1078 AAGCAGUAAUAUUUUAA AGATGGACTGGGAA GAUGGACUGGGAA LDHA_exon9 + ATTT 502 TAAGATGGACTGGGAA 1079 UAAGAUGGACUGGGAAA AAACATCAACTCCT AACAUCAACUCCU LDHA_exon9 + TTTT 503 AAGATGGACTGGGAAA 1080 AAGAUGGACUGGGAAAA AACATCAACTCCTG ACAUCAACUCCUG LDHA_exon9 + TTTA 504 AGATGGACTGGGAAAA 1081 AGAUGGACUGGGAAAAA ACATCAACTCCTGA CAUCAACUCCUGA LDHA_exon9 + GTTA 505 GAAATAAGAATGGTTT 1082 GAAAUAAGAAUGGUUUG GTAAAATCCACAGC UAAAAUCCACAGC LDHA_exon9 + GTTT 506 GTAAAATCCACAGCTA 1083 GUAAAAUCCACAGCUAU TATCCTGATGCTGG AUCCUGAUGCUGG LDHA_exon9 + TTTG 507 TAAAATCCACAGCTAT 1084 UAAAAUCCACAGCUAUA ATCCTGATGCTGGA UCCUGAUGCUGGA LDHA_exon9 + ATTA 508 ATCTTGTGTAGTCTTC 1085 AUCUUGUGUAGUCUUCA AACTGGTTAGTGTG ACUGGUUAGUGUG LDHA_exon9 + CTTG 509 TGTAGTCTTCAACTGG 1086 UGUAGUCUUCAACUGGU TTAGTGTGAAATAG UAGUGUGAAAUAG LDHA_exon9 + CTTC 510 AACTGGTTAGTGTGAA 1087 AACUGGUUAGUGUGAAA ATAGTTCTGCCACC UAGUUCUGCCACC LDHA_exon9 + GTTA 511 GTGTGAAATAGTTCTG 1088 GUGUGAAAUAGUUCUGC CCACCTCTGACGCA CACCUCUGACGCA LDHA_exon9 + GTTC 512 TGCCACCTCTGACGCA 1089 UGCCACCUCUGACGCAC CCACTGCCAATGCT CACUGCCAAUGCU LDHA_exon9 + ATTT 513 GCCCCTTGAGCCAGGT 1090 GCCCCUUGAGCCAGGUG GGATGTTTACCGTG GAUGUUUACCGUG LDHA_exon9 + TTTT 514 ATCTGATCTGTGATTA 1091 AUCUGAUCUGUGAUUAA AAGCAGTAATATTT AGCAGUAAUAUUU LDHA_exon9 − CTTC 515 CCAAAGTGCTGGGATT 1092 CCAAAGUGCUGGGAUUA ATAGGCATGAGCCA UAGGCAUGAGCCA LDHA_exon9 + CTTG 516 GGCAACCCTGCAACGA 1093 GGCAACCCUGCAACGAU TTTTTTCTAACAGG UUUUUCUAACAGG LDHA_exon9 + TTTT 517 TTCTAACAGGGATATT 1094 UUCUAACAGGGAUAUUA ATTGACTAATAGCA UUGACUAAUAGCA LDHA_exon9 − ATTT 518 TCAGAAAAATGTGCAG 1095 UCAGAAAAAUGUGCAGA AAAACTTGAGTAGA AAACUUGAGUAGA LDHA_exon9 − TTTT 519 CAGAAAAATGTGCAGA 1096 CAGAAAAAUGUGCAGAA AAACTTGAGTAGAC AACUUGAGUAGAC LDHA_exon9 − TTTC 520 AGAAAAATGTGCAGAA 1097 AGAAAAAUGUGCAGAAA AACTTGAGTAGACA ACUUGAGUAGACA LDHA_exon9 − CTTG 521 AGTAGACATCCACCAA 1098 AGUAGACAUCCACCAAG GGTTACTTGTTTTT GUUACUUGUUUUU LDHA_exon9 − GTTA 522 CTTGTTTTTTTTGGTT 1099 CUUGUUUUUUUUGGUUU TTGTTTTGTTTTTT UGUUUUGUUUUUU LDHA_exon9 − CTTG 523 TTTTTTTTGGTTTTGT 1100 UUUUUUUUGGUUUUGUU TTTGTTTTTTTAAC UUGUUUUUUUAAC LDHA_exon9 − GTTT 524 TTTTTGGTTTTGTTTT 1101 UUUUUGGUUUUGUUUUG GTTTTTTTAACAGA UUUUUUUAACAGA LDHA_exon9 − TTTT 525 TTTTGGTTTTGTTTTG 1102 UUUUGGUUUUGUUUUGU TTTTTTTAACAGAT UUUUUUAACAGAU LDHA_exon9 − TTTT 526 TTTGGTTTTGTTTTGT 1103 UUUGGUUUUGUUUUGUU TTTTTTAACAGATG UUUUUAACAGAUG LDHA_exon9 − TTTT 527 TTGGTTTTGTTTTGTT 1104 UUGGUUUUGUUUUGUUU TTTTTAACAGATGG UUUUAACAGAUGG LDHA_exon9 − TTTT 528 TGGTTTTGTTTTGTTT 1105 UGGUUUUGUUUUGUUUU TTTTAACAGATGGG UUUAACAGAUGGG LDHA_exon9 − TTTT 529 GGTTTTGTTTTGTTTT 1106 GGUUUUGUUUUGUUUUU TTTAACAGATGGGG UUAACAGAUGGGG LDHA_exon9 − TTTG 530 GTTTTGTTTTGTTTTT 1107 GUUUUGUUUUGUUUUUU TTAACAGATGGGGT UAACAGAUGGGGU LDHA_exon9 − GTTT 531 TGTTTTGTTTTTTTAA 1108 UGUUUUGUUUUUUUAAC CAGATGGGGTTTTG AGAUGGGGUUUUG LDHA_exon9 − GTTG 532 TATTTTCAGAAAAATG 1109 UAUUUUCAGAAAAAUGU TGCAGAAAACTTGA GCAGAAAACUUGA LDHA_exon9 − TTTT 533 GTTTTGTTTTTTTAAC 1110 GUUUUGUUUUUUUAACA AGATGGGGTTTTGT GAUGGGGUUUUGU LDHA_exon9 − GTTT 534 TGTTTTTTTAACAGAT 1111 UGUUUUUUUAACAGAUG GGGGTTTTGTTGTG GGGUUUUGUUGUG LDHA_exon9 − TTTT 535 GTTTTTTTAACAGATG 1112 GUUUUUUUAACAGAUGG GGGTTTTGTTGTGT GGUUUUGUUGUGU LDHA_exon9 − TTTG 536 TTTTTTTAACAGATGG 1113 UUUUUUUAACAGAUGGG GGTTTTGTTGTGTT GUUUUGUUGUGUU LDHA_exon9 − GTTT 537 TTTTAACAGATGGGGT 1114 UUUUAACAGAUGGGGUU TTTGTTGTGTTGGC UUGUUGUGUUGGC LDHA_exon9 − TTTT 538 TTTAACAGATGGGGTT 1115 UUUAACAGAUGGGGUUU TTGTTGTGTTGGCC UGUUGUGUUGGCC LDHA_exon9 − TTTT 539 TTAACAGATGGGGTTT 1116 UUAACAGAUGGGGUUUU TGTTGTGTTGGCCA GUUGUGUUGGCCA LDHA_exon9 − TTTT 540 TAACAGATGGGGTTTT 1117 UAACAGAUGGGGUUUUG GTTGTGTTGGCCAG UUGUGUUGGCCAG LDHA_exon9 − TTTT 541 AACAGATGGGGTTTTG 1118 AACAGAUGGGGUUUUGU TTGTGTTGGCCAGG UGUGUUGGCCAGG LDHA_exon9 − TTTA 542 ACAGATGGGGTTTTGT 1119 ACAGAUGGGGUUUUGUU TGTGTTGGCCAGGC GUGUUGGCCAGGC LDHA_exon9 − GTTT 543 TGTTGTGTTGGCCAGG 1120 UGUUGUGUUGGCCAGGC CTGGTCCCCAATTC UGGUCCCCAAUUC LDHA_exon9 − TTTT 544 GTTGTGTTGGCCAGGC 1121 GUUGUGUUGGCCAGGCU TGGTCCCCAATTCC GGUCCCCAAUUCC LDHA_exon9 − TTTG 545 TTGTGTTGGCCAGGCT 1122 UUGUGUUGGCCAGGCUG GGTCCCCAATTCCT GUCCCCAAUUCCU LDHA_exon9 − GTTG 546 TGTTGGCCAGGCTGGT 1123 UGUUGGCCAGGCUGGUC CCCCAATTCCTGGC CCCAAUUCCUGGC LDHA_exon9 − GTTG 547 GCCAGGCTGGTCCCCA 1124 GCCAGGCUGGUCCCCAA ATTCCTGGCCTCCA UUCCUGGCCUCCA LDHA_exon9 − TTTG 548 TTTTGTTTTTTTAACA 1125 UUUUGUUUUUUUAACAG GATGGGGTTTTGTT AUGGGGUUUUGUU LDHA_exon9 + ATTT 549 TTTCTAACAGGGATAT 1126 UUUCUAACAGGGAUAUU TATTGACTAATAGC AUUGACUAAUAGC LDHA_exon9 + TTTG 550 TGCACATTTTTCTGAA 1127 UGCACAUUUUUCUGAAA AATACAACTGTGAC AUACAACUGUGAC LDHA_exon9 + GTTT 551 TCTGCACATTTTTCTG 1128 UCUGCACAUUUUUCUGA AAAATACAACTGTG AAAUACAACUGUG LDHA_exon9 + TTTT 552 TCTAACAGGGATATTA 1129 UCUAACAGGGAUAUUAU TTGACTAATAGCAG UGACUAAUAGCAG LDHA_exon9 + TTTT 553 CTAACAGGGATATTAT 1130 CUAACAGGGAUAUUAUU TGACTAATAGCAGA GACUAAUAGCAGA LDHA_exon9 + TTTG 554 TAACAGGGATATTATT 1131 UAACAGGGAUAUUAUUG GACTAATAGCAGAG ACUAAUAGCAGAG LDHA_exon9 + ATTA 555 TTGACTAATAGCAGAG 1132 UUGACUAAUAGCAGAGG GATGTAATAGTCAA AUGUAAUAGUCAA LDHA_exon9 + ATTG 556 ACTAATAGCAGAGGAT 1133 ACUAAUAGCAGAGGAUG GTAATAGTCAACTG UAAUAGUCAACUG LDHA_exon9 + GTTG 557 TATTGGTACCACTTCC 1134 UAUUGGUACCACUUCCA ATTGTAAGTCCCAA UUGUAAGUCCCAA LDHA_exon9 + ATTG 558 GTACCACTTCCATTGT 1135 GUACCACUUCCAUUGUA AAGTCCCAAAGTAT AGUCCCAAAGUAU LDHA_exon9 + CTTC 559 CATTGTAAGTCCCAAA 1136 CAUUGUAAGUCCCAAAG GTATTATATATTTG UAUUAUAUAUUUG LDHA_exon9 + ATTG 560 TAAGTCCCAAAGTATT 1137 UAAGUCCCAAAGUAUUA ATATATTTGATAAT UAUAUUUGAUAAU LDHA_exon9 + ATTA 561 TATATTTGATAATAAT 1138 UAUAUUUGAUAAUAAUG GCTAATCATAATTG CUAAUCAUAAUUG LDHA_exon9 + ATTT 562 GATAATAATGCTAATC 1139 GAUAAUAAUGCUAAUCA ATAATTGGAAAGTA UAAUUGGAAAGUA LDHA_exon9 + TTTG 563 ATAATAATGCTAATCA 1140 AUAAUAAUGCUAAUCAU TAATTGGAAAGTAA AAUUGGAAAGUAA LDHA_exon9 + ATTG 564 GAAAGTAACATTCTAT 1141 GAAAGUAACAUUCUAUA ATGTAAATGTAAAA UGUAAAUGUAAAA LDHA_exon9 + ATTG 565 TATATGTAAATGTAAA 1142 UAUAUGUAAAUGUAAAA ATTTATTTGCCAAC UUUAUUUGCCAAC LDHA_exon9 + TTTT 566 CTGCACATTTTTCTGA 1143 CUGCACAUUUUUCUGAA AAATACAACTGTGA AAUACAACUGUGA LDHA_exon9 + ATTT 567 ATTTGCCAACTGAATA 1144 AUUUGCCAACUGAAUAU TAGGCAATGATAGT AGGCAAUGAUAGU LDHA_exon9 + ATTT 568 GCCAACTGAATATAGG 1145 GCCAACUGAAUAUAGGC CAATGATAGTGTGT AAUGAUAGUGUGU LDHA_exon9 + TTTG 569 CCAACTGAATATAGGC 1146 CCAACUGAAUAUAGGCA AATGATAGTGTGTC AUGAUAGUGUGUC LDHA_exon9 + ATTT 570 TTGAGATCTTGTCCTC 1147 UUGAGAUCUUGUCCUCU TGGAAGCTGGTAAC GGAAGCUGGUAAC LDHA_exon9 + TTTT 571 TGAGATCTTGTCCTCT 1148 UGAGAUCUUGUCCUCUG GGAAGCTGGTAACA GAAGCUGGUAACA LDHA_exon9 + TTTT 572 GAGATCTTGTCCTCTG 1149 GAGAUCUUGUCCUCUGG GAAGCTGGTAACAA AAGCUGGUAACAA LDHA_exon9 + TTTG 573 AGATCTTGTCCTCTGG 1150 AGAUCUUGUCCUCUGGA AAGCTGGTAACAAT AGCUGGUAACAAU LDHA_exon9 + GTTG 574 TCCTCTGGAAGCTGGT 1151 UCCUCUGGAAGCUGGUA AACAATTAAAAACA ACAAUUAAAAACA LDHA_exon9 + ATTA 575 AAAACAATCTTAAGGC 1152 AAAACAAUCUUAAGGCA AGGGTGCAGTGGCT GGGUGCAGUGGCU LDHA_exon9 + CTTA 576 AGGCAGGGTGCAGTGG 1153 AGGCAGGGUGCAGUGGC CTCATGCCTATAAT UCAUGCCUAUAAU LDHA_exon9 + CTTT 577 GGGAAGCCCAGGTGGG 1154 GGGAAGCCCAGGUGGGC CTGATCACTGGAGG UGAUCACUGGAGG LDHA_exon9 + TTTG 578 GGAAGCCCAGGTGGGC 1155 GGAAGCCCAGGUGGGCU TGATCACTGGAGGC GAUCACUGGAGGC LDHA_exon9 + ATTG 579 GGGACCAGCCTGGCCA 1156 GGGACCAGCCUGGCCAA ACACAACAAAACCC CACAACAAAACCC LDHA_exon9 + GTTA 580 AAAAAACAAAACAAAA 1157 AAAAAACAAAACAAAAC CCAAAAAAAACAAG CAAAAAAAACAAG LDHA_exon9 + GTTG 581 GTGGATGTCTACTCAA 1158 GUGGAUGUCUACUCAAG GTTTTCTGCACATT UUUUCUGCACAUU LDHA_exon9 + TTTA 582 TTTGCCAACTGAATAT 1159 UUUGCCAACUGAAUAUA AGGCAATGATAGTG GGCAAUGAUAGUG LDHA_exon9 + CTTA 583 GTGTTCCTTGCATTTT 1160 GUGUUCCUUGCAUUUUG GGGACAGAATGGAA GGACAGAAUGGAA LDHA_exon9 + ATTT 584 TTCTGAAAATACAACT 1161 UUCUGAAAAUACAACUG GTGACCCTTA UGACCCUUA LDHA_exon9 + TTTT 585 TCTGAAAATACAACTG 1162 UCUGAAAAUACAACUGU TGACCCTTA GACCCUUA LDHA_exon9 + TTTT 586 CTGAAAATACAACTGT 1163 CUGAAAAUACAACUGUG GACCCTTA ACCCUUA LDHA_exon9 + TTTC 587 TGAAAATACAACTGTG 1164 UGAAAAUACAACUGUGA ACCCTTA CCCUUA *The 3′ three nucleotides represent the 5′-TTN-3′ motif.

The present disclosure includes all combinations of the direct repeats and spacers listed above, consistent with the disclosure herein.

In some embodiments, a spacer sequence described herein comprises a uracil (U). In some embodiments, a spacer sequence described herein comprises a thymine (T). In some embodiments, a spacer sequence according to Table 5 comprises a sequence comprising a thymine in one or more places indicated as uracil in Table 5.

(iii). Exemplary RNA Guides

The present disclosure includes RNA guides that comprise any and all combinations of the direct repeats and spacers described herein (e.g., as set forth in Table 5, above). In some embodiments, the sequence of an RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229. In some embodiments, an RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229.

In some embodiments, exemplary RNA guides provided herein may comprise a spacer sequence of any one of SEQ ID NOs: 1269-1273. In one example, the RNA guide may comprise a spacer of SEQ ID NO: 1272. In another example, the RNA guide may comprise a spacer of SEQ ID NO: 1269. In still another example, the RNA guide may comprise a spacer of SEQ ID NO: 1270. In still another example, the RNA guide may comprise a spacer of SEQ ID NO: 1271. In yet another example, the RNA guide may comprise a spacer of SEQ ID NO: 1273.

Any of the exemplary RNA guides disclosed herein may comprise a direct sequence of any one of SEQ ID NOs:1-10 or a fragment thereof that is at least 23-nucleotide in length. In one example, the direct sequence may comprise SEQ ID NO: 10.

In specific examples, the RNA guides provide herein may comprise the nucleotide sequence of SEQ ID NOs: 1214, 1235, 1221, 1224 or 1225. In one example, the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1224. In another example, the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1214. In still another example, the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1235. In still another example, the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1221. In yet another example, the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1225.

(iv). Modifications

The RNA guide may include one or more covalent modifications with respect to a reference sequence, in particular the parent polyribonucleotide, which are included within the scope of this invention.

Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below.

The RNA guide may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.

In some embodiments, the modification may include a chemical or cellular induced modification. For example, some nonlimiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to RNA guide-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.

Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) may exist at various positions in the sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the function of the sequence is not substantially decreased. The sequence may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).

In some embodiments, sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar at one or more ribonucleotides of the sequence may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, a sequence will include ribonucleotides with a phosphorus atom in its internucleoside backbone.

Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. In some embodiments, the sequence may be negatively or positively charged.

The modified nucleotides, which may be incorporated into the sequence, can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

The α-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.

In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine (α-thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-pseudouridine).

Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.

In some embodiments, the sequence may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4′-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2′-deoxy-2′-methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1-beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5′-elaidic acid ester).

In some embodiments, the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197) In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In some embodiments, mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

The sequence may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally-occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the sequence, or in a given predetermined sequence region thereof. In some embodiments, the sequence includes a pseudouridine. In some embodiments, the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.

In some embodiments, one or more of the nucleotides of an RNA guide comprises a 2′-O-methyl phosphorothioate modification. In some embodiments, each of the first three nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification. In some embodiments, each of the last four nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification. In some embodiments, each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification, and wherein the last nucleotide of the RNA guide is unmodified. In some embodiments, each of the first three nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification, and each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification.

When a gene editing system disclosed herein comprises nucleic acids encoding the Cas12i polypeptide disclosed herein, e.g., mRNA molecules, such nucleic acid molecules may contain any of the modifications disclosed herein, where applicable.

B. Cas12i Polypeptide

In some embodiments, the composition or system of the present disclosure includes a Cas12i polypeptide as described in WO/2019/178427, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.

In some embodiments, the composition of the present disclosure includes a Cas12i2 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1166 and/or encoded by SEQ ID NO: 1165). In some embodiments, the Cas12i2 polypeptide comprises at least one RuvC domain.

A nucleic acid sequence encoding the Cas12i2 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1165. In some embodiments, the Cas12i2 polypeptide is encoded by a nucleic acid comprising a sequence having least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the reference nucleic acid sequence, e.g., SEQ ID NO: 1165. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions of temperature and ionic strength (e.g., within a range of medium to high stringency). See, e.g., Tijssen, “Hybridization with Nucleic Acid Probes. Part I. Theory and Nucleic Acid Preparation” (Laboratory Techniques in Biochemistry and Molecular Biology, Vol 24).

In some embodiments, the Cas12i2 polypeptide is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity, but not 100% sequence identity, to a reference nucleic acid sequence, e.g., SEQ ID NO: 1165.

In some embodiments, the Cas12i2 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1166.

In some embodiments, the present disclosure describes a Cas12i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1166. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i2 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1166 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

In some embodiments, the Cas12i2 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171. In specific examples, the Cas12i2 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1168 or SEQ ID NO: 1171.

In some examples, the Cas12i2 polypeptide may contain one or more mutations relative to SEQ ID NO: 1166, for example, at position D581, G624, F626, P868, I926, V1030, E1035, S1046, or any combination thereof. In some instances, the one or more mutations are amino acid substitutions, for example, D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.

In some examples, the Cas12i2 polypeptide contains mutations at positions D581, D911, 1926, and V1030. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, D911R, I926R, and V1030G (e.g., SEQ ID NO: 1167). In some examples, the Cas12i2 polypeptide contains mutations at positions D581, I926, and V1030. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, I926R, and V1030G (e.g., SEQ ID NO: 1168). In some examples, the Cas12i2 polypeptide may contain mutations at positions D581, I926, V1030, and S1046. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, I926R, V1030G, and S1046G (e.g., SEQ ID NO: 1169). In some examples, the Cas12i2 polypeptide may contain mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G (e.g., SEQ ID NO: 1170). In some examples, the Cas12i2 polypeptide may contain mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G (e.g., SEQ ID NO: 1171).

In some embodiments, the Cas12i2 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171. In some embodiments, a Cas12i2 polypeptide having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171 maintains the amino acid changes (or at least 1, 2, 3 etc. of these changes) that differentiate the polypeptide from its respective parent/reference sequence.

In some embodiments, the present disclosure describes a Cas12i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i2 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

In some embodiments, the composition of the present disclosure includes a Cas12i4 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1202 and/or encoded by SEQ ID NO: 1201). In some embodiments, the Cas12i4 polypeptide comprises at least one RuvC domain.

A nucleic acid sequence encoding the Cas12i4 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1201. In some embodiments, the Cas12i4 polypeptide is encoded by a nucleic acid comprising a sequence having least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the reference nucleic acid sequence, e.g., SEQ ID NO: 1201. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions of temperature and ionic strength (e.g., within a range of medium to high stringency).

In some embodiments, the Cas12i4 polypeptide is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity, but not 100% sequence identity, to a reference nucleic acid sequence, e.g., SEQ ID NO: 1201.

In some embodiments, the Cas12i4 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1202.

In some embodiments, the present disclosure describes a Cas12i4 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1202. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i4 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1202 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

In some embodiments, the Cas12i4 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1203 or SEQ ID NO: 1204.

In some embodiments, the Cas12i4 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1203 or SEQ ID NO: 1204. In some embodiments, a Cas12i4 polypeptide having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1203 or SEQ ID NO: 1204 maintains the amino acid changes (or at least 1, 2, 3 etc. of these changes) that differentiate it from its respective parent/reference sequence.

In some embodiments, the present disclosure describes a Cas12i4 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1203 or SEQ ID NO: 1204. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i4 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1203 or SEQ ID NO: 1204 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

In some embodiments, the composition of the present disclosure includes a Cas12i1 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1211). In some embodiments, the Cas12i4 polypeptide comprises at least one RuvC domain.

In some embodiments, the Cas12i1 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1211.

In some embodiments, the present disclosure describes a Cas12i1 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1211. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i1 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1211 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

In some embodiments, the composition of the present disclosure includes a Cas12i3 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1212). In some embodiments, the Cas12i4 polypeptide comprises at least one RuvC domain.

In some embodiments, the Cas12i3 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1212.

In some embodiments, the present disclosure describes a Cas12i3 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1212. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i3 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1212 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

Although the changes described herein may be one or more amino acid changes, changes to the Cas12i polypeptide may also be of a substantive nature, such as fusion of polypeptides as amino- and/or carboxyl-terminal extensions. For example, the Cas12i polypeptide may contain additional peptides, e.g., one or more peptides. Examples of additional peptides may include epitope peptides for labelling, such as a polyhistidine tag (His-tag), Myc, and FLAG. In some embodiments, the Cas12i polypeptide described herein can be fused to a detectable moiety such as a fluorescent protein (e.g., green fluorescent protein (GFP) or yellow fluorescent protein (YFP)).

In some embodiments, the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.

In some embodiments, the Cas12i polypeptide described herein can be self-inactivating. See, Epstein et al., “Engineering a Self-Inactivating CRISPR System for AAV Vectors,” Mol. Ther., 24 (2016): S50, which is incorporated by reference in its entirety.

In some embodiments, the nucleotide sequence encoding the Cas12i polypeptide described herein can be codon-optimized for use in a particular host cell or organism. For example, the nucleic acid can be codon-optimized for any non-human eukaryote including mice, rats, rabbits, dogs, livestock, or non-human primates. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.). In some examples, the nucleic acid encoding the Cas12i polypeptides such as Cas12i2 polypeptides as disclosed herein can be an mRNA molecule, which can be codon optimized.

Exemplary Cas12i polypeptide sequences and corresponding nucleotide sequences are listed in Table 6.

TABLE 6 Cas12i and LDHA Sequences SEQ ID Description NO Sequence 1165 ATGAGCAGCGCGATCAAAAGCTACAAGAGCGTTCTGCGTCCGAACGAGCGTAAGAA Nucleotide CCAACTGCTGAAAAGCACCATTCAGTGCCTGGAAGACGGTAGCGCGTTCTTTTTCA sequence AGATGCTGCAAGGCCTGTTTGGTGGCATCACCCCGGAGATTGTTCGTTTCAGCACC encoding GAACAGGAGAAACAGCAACAGGATATCGCGCTGTGGTGCGCGGTTAACTGGTTCCG parent TCCGGTGAGCCAAGACAGCCTGACCCACACCATTGCGAGCGATAACCTGGTGGAGA Cas12i2 AGTTTGAGGAATACTATGGTGGCACCGCGAGCGACGCGATCAAACAGTACTTCAGC GCGAGCATTGGCGAAAGCTACTATTGGAACGACTGCCGTCAACAGTACTATGATCT GTGCCGTGAGCTGGGTGTTGAGGTGAGCGACCTGACCCATGATCTGGAGATCCTGT GCCGTGAAAAGTGCCTGGCGGTTGCGACCGAGAGCAACCAGAACAACAGCATCATT AGCGTTCTGTTTGGCACCGGCGAAAAAGAGGACCGTAGCGTGAAACTGCGTATCAC CAAGAAAATTCTGGAGGCGATCAGCAACCTGAAAGAAATCCCGAAGAACGTTGCGC CGATTCAAGAGATCATTCTGAACGTGGCGAAAGCGACCAAGGAAACCTTCCGTCAG GTGTATGCGGGTAACCTGGGTGCGCCGAGCACCCTGGAGAAATTTATCGCGAAGGA CGGCCAAAAAGAGTTCGATCTGAAGAAACTGCAGACCGACCTGAAGAAAGTTATTC GTGGTAAAAGCAAGGAGCGTGATTGGTGCTGCCAGGAAGAGCTGCGTAGCTACGTG GAGCAAAACACCATCCAGTATGACCTGTGGGCGTGGGGCGAAATGTTCAACAAAGC GCACACCGCGCTGAAAATCAAGAGCACCCGTAACTACAACTTTGCGAAGCAACGTC TGGAACAGTTCAAAGAGATTCAGAGCCTGAACAACCTGCTGGTTGTGAAGAAGCTG AACGACTTTTTCGATAGCGAATTTTTCAGCGGCGAGGAAACCTACACCATCTGCGT TCACCATCTGGGTGGCAAGGACCTGAGCAAACTGTATAAGGCGTGGGAGGATGATC CGGCGGACCCGGAAAACGCGATTGTGGTTCTGTGCGACGATCTGAAAAACAACTTT AAGAAAGAGCCGATCCGTAACATTCTGCGTTACATCTTCACCATTCGTCAAGAATG CAGCGCGCAGGACATCCTGGCGGCGGCGAAGTACAACCAACAGCTGGATCGTTATA AAAGCCAAAAGGCGAACCCGAGCGTTCTGGGTAACCAGGGCTTTACCTGGACCAAC GCGGTGATCCTGCCGGAGAAGGCGCAGCGTAACGACCGTCCGAACAGCCTGGATCT GCGTATTTGGCTGTACCTGAAACTGCGTCACCCGGACGGTCGTTGGAAGAAACACC ATATCCCGTTCTACGATACCCGTTTCTTCCAAGAAATTTATGCGGCGGGCAACAGC CCGGTTGACACCTGCCAGTTTCGTACCCCGCGTTTCGGTTATCACCTGCCGAAACT GACCGATCAGACCGCGATCCGTGTTAACAAGAAACATGTGAAAGCGGCGAAGACCG AGGCGCGTATTCGTCTGGCGATCCAACAGGGCACCCTGCCGGTGAGCAACCTGAAG ATCACCGAAATTAGCGCGACCATCAACAGCAAAGGTCAAGTGCGTATTCCGGTTAA GTTTGACGTGGGTCGTCAAAAAGGCACCCTGCAGATCGGTGACCGTTTCTGCGGCT ACGATCAAAACCAGACCGCGAGCCACGCGTATAGCCTGTGGGAAGTGGTTAAAGAG GGTCAATACCATAAAGAGCTGGGCTGCTTTGTTCGTTTCATCAGCAGCGGTGACAT CGTGAGCATTACCGAGAACCGTGGCAACCAATTTGATCAGCTGAGCTATGAAGGTC TGGCGTACCCGCAATATGCGGACTGGCGTAAGAAAGCGAGCAAGTTCGTGAGCCTG TGGCAGATCACCAAGAAAAACAAGAAAAAGGAAATCGTGACCGTTGAAGCGAAAGA GAAGTTTGACGCGATCTGCAAGTACCAGCCGCGTCTGTATAAATTCAACAAGGAGT ACGCGTATCTGCTGCGTGATATTGTTCGTGGCAAAAGCCTGGTGGAACTGCAACAG ATTCGTCAAGAGATCTTTCGTTTCATTGAACAGGACTGCGGTGTTACCCGTCTGGG CAGCCTGAGCCTGAGCACCCTGGAAACCGTGAAAGCGGTTAAGGGTATCATTTACA GCTATTTTAGCACCGCGCTGAACGCGAGCAAGAACAACCCGATCAGCGACGAACAG CGTAAAGAGTTTGATCCGGAACTGTTCGCGCTGCTGGAAAAGCTGGAGCTGATTCG TACCCGTAAAAAGAAACAAAAAGTGGAACGTATCGCGAACAGCCTGATTCAGACCT GCCTGGAGAACAACATCAAGTTCATTCGTGGTGAAGGCGACCTGAGCACCACCAAC AACGCGACCAAGAAAAAGGCGAACAGCCGTAGCATGGATTGGTTGGCGCGTGGTGT TTTTAACAAAATCCGTCAACTGGCGCCGATGCACAACATTACCCTGTTCGGTTGCG GCAGCCTGTACACCAGCCACCAGGACCCGCTGGTGCATCGTAACCCGGATAAAGCG ATGAAGTGCCGTTGGGCGGCGATCCCGGTTAAGGACATTGGCGATTGGGTGCTGCG TAAGCTGAGCCAAAACCTGCGTGCGAAAAACATCGGCACCGGCGAGTACTATCACC AAGGTGTTAAAGAGTTCCTGAGCCATTATGAACTGCAGGACCTGGAGGAAGAGCTG CTGAAGTGGCGTAGCGATCGTAAAAGCAACATTCCGTGCTGGGTGCTGCAGAACCG TCTGGCGGAGAAGCTGGGCAACAAAGAAGCGGTGGTTTACATCCCGGTTCGTGGTG GCCGTATTTATTTTGCGACCCACAAGGTGGCGACCGGTGCGGTGAGCATCGTTTTC GACCAAAAACAAGTGTGGGTTTGCAACGCGGATCATGTTGCGGCGGCGAACATCGC GCTGACCGTGAAGGGTATTGGCGAACAAAGCAGCGACGAAGAGAACCCGGATGGTA GCCGTATCAAACTGCAGCTGACCAGC 1166 MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFST Parent EQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFS Cas12i2 ASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSII amino acid SVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQ sequence VYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYV EQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKL NDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNF KKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTN AVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNS PVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLK ITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKE GQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSL WQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQ IRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQ RKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTN NATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKA MKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGTGEYYHQGVKEFLSHYELQDLEEEL LKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVF DQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS 1167 MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE Variant IVRFSTEQEK QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG Cas12i2 of GTASDAIKQY FSASIGESYY WNDCRQQYYD LCRELGVEVS DLTHDLEILC SEQ ID REKCLAVATE SNQNNSIISV LFGTGEKEDR SVKLRITKKI LEAISNLKEI NO: 3 of PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI AKDGQKEFDL PCT/US20 KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMFNKAH 21/025257 TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET YTICVHHLGG KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI LRYIFTIRQE CSAQDILAAA KYNQQLDRYK SQKANPSVLG NQGFTWTNAV ILPEKAQRND RPNSLDLRIW LYLKLRHPDG RWKKHHIPFY DTRFFQEIYA AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK TEARIRLAIQ QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ NQTASHAYSL WEVVKEGQYH KELGCFVRFI SSGDIVSITE NRGNQFDQLS YEGLAYPQYA DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ PRLYKFNKEY AYLLRDIVRG KSLVELQQIR QEIFRFIEQD CGVTRLGSLS LSTLETVKAV KGIIYSYFST ALNASKNNPI SDEQRKEFDP ELFALLEKLE LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN NATKKKANSR SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR WAAIPVKDIG RWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE ELLKWRSDRK SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA TGAVSIVFDQ KQVWVCNADH VAAANIALTG KGIGEQSSDE ENPDGSRIKL QLTS 1168 MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE Variant IVRFSTEQEK QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG Cas12i2 of GTASDAIKQY FSASIGESYY WNDCRQQYYD LCRELGVEVS DLTHDLEILC SEQ ID REKCLAVATE SNQNNSIISV LFGTGEKEDR SVKLRITKKI LEAISNLKEI NO: 4 of PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI AKDGQKEFDL PCT/US20 KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMFNKAH 21/025257 TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET YTICVHHLGG KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI LRYIFTIRQE CSAQDILAAA KYNQQLDRYK SQKANPSVLG NQGFTWTNAV ILPEKAQRND RPNSLDLRIW LYLKLRHPDG RWKKHHIPFY DTRFFQEIYA AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK TEARIRLAIQ QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ NQTASHAYSL WEVVKEGQYH KELGCFVRFI SSGDIVSITE NRGNQFDQLS YEGLAYPQYA DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ PRLYKFNKEY AYLLRDIVRG KSLVELQQIR QEIFRFIEQD CGVTRLGSLS LSTLETVKAV KGIIYSYFST ALNASKNNPI SDEQRKEFDP ELFALLEKLE LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN NATKKKANSR SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE ELLKWRSDRK SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA TGAVSIVFDQ KQVWVCNADH VAAANIALTG KGIGEQSSDE ENPDGSRIKL QLTS 1169 MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE Variant IVRFSTEQEK QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG Cas12i2 of GTASDAIKQY FSASIGESYY WNDCRQQYYD LCRELGVEVS DLTHDLEILC SEQ ID REKCLAVATE SNQNNSIISV LFGTGEKEDR SVKLRITKKI LEAISNLKEI NO: 5 of PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI AKDGQKEFDL PCT/US20 KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMFNKAH 21/025257 TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET YTICVHHLGG KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI LRYIFTIRQE CSAQDILAAA KYNQQLDRYK SQKANPSVLG NQGFTWTNAV ILPEKAQRND RPNSLDLRIW LYLKLRHPDG RWKKHHIPFY DTRFFQEIYA AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK TEARIRLAIQ QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ NQTASHAYSL WEVVKEGQYH KELGCFVRFI SSGDIVSITE NRGNQFDQLS YEGLAYPQYA DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ PRLYKFNKEY AYLLRDIVRG KSLVELQQIR QEIFRFIEQD CGVTRLGSLS LSTLETVKAV KGIIYSYFST ALNASKNNPI SDEQRKEFDP ELFALLEKLE LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN NATKKKANSR SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE ELLKWRSDRK SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA TGAVSIVFDQ KQVWVCNADH VAAANIALTG KGIGEQSSDE ENPDGGRIKL QLTS 1170 MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE Variant IVRFSTEQEK QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG Cas12i2 of GTASDAIKQY FSASIGESYY WNDCRQQYYD LCRELGVEVS DLTHDLEILC SEQ ID REKCLAVATE SNQNNSIISV LFGTGEKEDR SVKLRITKKI LEAISNLKEI NO: 495 of PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI AKDGQKEFDL PCT/US20 KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMFNKAH 21/025257 TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET YTICVHHLGG KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI LRYIFTIRQE CSAQDILAAA KYNQQLDRYK SQKANPSVLG NQGFTWTNAV ILPEKAQRND RPNSLDLRIW LYLKLRHPDG RWKKHHIPFY DTRFFQEIYA AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK TEARIRLAIQ QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ NQTASHAYSL WEVVKEGQYH KELRCRVRFI SSGDIVSITE NRGNQFDQLS YEGLAYPQYA DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ PRLYKFNKEY AYLLRDIVRG KSLVELQQIR QEIFRFIEQD CGVTRLGSLS LSTLETVKAV KGIIYSYFST ALNASKNNPI SDEQRKEFDP ELFALLEKLE LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN NATKKKANSR SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE ELLKWRSDRK SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA TGAVSIVFDQ KQVWVCNADH VAAANIALTG KGIGRQSSDE ENPDGGRIKL QLTS 1171 MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE Variant IVRFSTEQEK QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG Cas12i2 of GTASDAIKQY FSASIGESYY WNDCRQQYYD LCRELGVEVS DLTHDLEILC SEQ ID REKCLAVATE SNQNNSIISV LFGTGEKEDR SVKLRITKKI LEAISNLKEI NO: 496 of PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI AKDGQKEFDL PCT/US20 KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMFNKAH 21/025257 TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET YTICVHHLGG KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI LRYIFTIRQE CSAQDILAAA KYNQQLDRYK SQKANPSVLG NQGFTWTNAV ILPEKAQRND RPNSLDLRIW LYLKLRHPDG RWKKHHIPFY DTRFFQEIYA AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK TEARIRLAIQ QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ NQTASHAYSL WEVVKEGQYH KELRCRVRFI SSGDIVSITE NRGNQFDQLS YEGLAYPQYA DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ PRLYKFNKEY AYLLRDIVRG KSLVELQQIR QEIFRFIEQD CGVTRLGSLS LSTLETVKAV KGIIYSYFST ALNASKNNPI SDEQRKEFDP ELFALLEKLE LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN NATKKKANSR SMDWLARGVF NKIRQLATMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE ELLKWRSDRK SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA TGAVSIVFDQ KQVWVCNADH VAAANIALTG KGIGRQSSDE ENPDGGRIKL QLTS 1201 ATGGCTTCCATCTCTAGGCCATACGGCACCAAGCTGCGACCGGACGCACGGAAGAA Nucleotide GGAGATGCTCGATAAGTTCTTTAATACACTGACTAAGGGTCAGCGCGTGTTCGCAG sequence ACCTGGCCCTGTGCATCTATGGCTCCCTGACCCTGGAGATGGCCAAGTCTCTGGAG encoding CCAGAAAGTGATTCAGAACTGGTGTGCGCTATTGGGTGGTTTCGGCTGGTGGACAA parent GACCATCTGGTCCAAGGATGGCATCAAGCAGGAGAATCTGGTGAAACAGTACGAAG Cas12i4 CCTATTCCGGAAAGGAGGCTTCTGAAGTGGTCAAAACATACCTGAACAGCCCCAGC TCCGACAAGTACGTGTGGATCGATTGCAGGCAGAAATTCCTGAGGTTTCAGCGCGA GCTCGGCACTCGCAACCTGTCCGAGGACTTCGAATGTATGCTCTTTGAACAGTACA TTAGACTGACCAAGGGCGAGATCGAAGGGTATGCCGCTATTTCAAATATGTTCGGA AACGGCGAGAAGGAAGACCGGAGCAAGAAAAGAATGTACGCTACACGGATGAAAGA TTGGCTGGAGGCAAACGAAAATATCACTTGGGAGCAGTATAGAGAGGCCCTGAAGA ACCAGCTGAATGCTAAAAACCTGGAGCAGGTTGTGGCCAATTACAAGGGGAACGCT GGCGGGGCAGACCCCTTCTTTAAGTATAGCTTCTCCAAAGAGGGAATGGTGAGCAA GAAAGAACATGCACAGCAGCTCGACAAGTTCAAAACCGTCCTGAAGAACAAAGCCC GGGACCTGAATTTTCCAAACAAGGAGAAGCTGAAGCAGTACCTGGAGGCCGAAATC GGCATTCCGGTCGACGCTAACGTGTACTCCCAGATGTTCTCTAACGGGGTGAGTGA GGTCCAGCCTAAGACCACACGGAATATGTCTTTTAGTAACGAGAAACTGGATCTGC TCACTGAACTGAAGGACCTGAACAAGGGCGATGGGTTCGAGTACGCCAGAGAAGTG CTGAACGGGTTCTTTGACTCCGAGCTCCACACTACCGAGGATAAGTTTAATATCAC CTCTAGGTACCTGGGAGGCGACAAATCAAACCGCCTGAGCAAACTCTATAAGATCT GGAAGAAAGAGGGTGTGGACTGCGAGGAAGGCATTCAGCAGTTCTGTGAAGCCGTC AAAGATAAGATGGGCCAGATCCCCATTCGAAATGTGCTGAAGTACCTGTGGCAGTT CCGGGAGACAGTCAGTGCCGAGGATTTTGAAGCAGCCGCTAAGGCTAACCATCTGG AGGAAAAGATCAGCCGGGTGAAAGCCCACCCAATCGTGATTAGCAATAGGTACTGG GCTTTTGGGACTTCCGCACTGGTGGGAAACATTATGCCCGCAGACAAGAGGCATCA GGGAGAGTATGCCGGTCAGAATTTCAAAATGTGGCTGGAGGCTGAACTGCACTACG ATGGCAAGAAAGCAAAGCACCATCTGCCTTTTTATAACGCCCGCTTCTTTGAGGAA GTGTACTGCTATCACCCCTCTGTCGCCGAGATCACTCCTTTCAAAACCAAGCAGTT TGGCTGTGAAATCGGGAAGGACATTCCAGATTACGTGAGCGTCGCTCTGAAGGACA ATCCGTATAAGAAAGCAACCAAACGAATCCTGCGTGCAATCTACAATCCCGTCGCC AACACAACTGGCGTTGATAAGACCACAAACTGCAGCTTCATGATCAAACGCGAGAA TGACGAATATAAGCTGGTCATCAACCGAAAAATTTCCGTGGATCGGCCTAAGAGAA TCGAAGTGGGCAGGACAATTATGGGGTACGACCGCAATCAGACAGCTAGCGATACT TATTGGATTGGCCGGCTGGTGCCACCTGGAACCCGGGGCGCATACCGCATCGGAGA GTGGAGCGTCCAGTATATTAAGTCCGGGCCTGTCCTGTCTAGTACTCAGGGAGTTA ACAATTCCACTACCGACCAGCTGGTGTACAACGGCATGCCATCAAGCTCCGAGCGG TTCAAGGCCTGGAAGAAAGCCAGAATGGCTTTTATCCGAAAACTCATTCGTCAGCT GAATGACGAGGGACTGGAATCTAAGGGTCAGGATTATATCCCCGAGAACCCTTCTA GTTTCGATGTGCGGGGCGAAACCCTGTACGTCTTTAACAGTAATTATCTGAAGGCC CTGGTGAGCAAACACAGAAAGGCCAAGAAACCTGTTGAGGGGATCCTGGACGAGAT TGAAGCCTGGACATCTAAAGACAAGGATTCATGCAGCCTGATGCGGCTGAGCAGCC TGAGCGATGCTTCCATGCAGGGAATCGCCAGCCTGAAGAGTCTGATTAACAGCTAC TTCAACAAGAATGGCTGTAAAACCATCGAGGACAAAGAAAAGTTTAATCCCGTGCT GTATGCCAAGCTGGTTGAGGTGGAACAGCGGAGAACAAACAAGCGGTCTGAGAAAG TGGGAAGAATCGCAGGTAGTCTGGAGCAGCTGGCCCTGCTGAACGGGGTTGAGGTG GTCATCGGCGAAGCTGACCTGGGGGAGGTCGAAAAAGGAAAGAGTAAGAAACAGAA TTCACGGAACATGGATTGGTGCGCAAAGCAGGTGGCACAGCGGCTGGAGTACAAAC TGGCCTTCCATGGAATCGGTTACTTTGGAGTGAACCCCATGTATACCAGCCACCAG GACCCTTTCGAACATAGGCGCGTGGCTGATCACATCGTCATGCGAGCACGTTTTGA GGAAGTCAACGTGGAGAACATTGCCGAATGGCACGTGCGAAATTTCTCAAACTACC TGCGTGCAGACAGCGGCACTGGGCTGTACTATAAGCAGGCCACCATGGACTTCCTG AAACATTACGGTCTGGAGGAACACGCTGAGGGCCTGGAAAATAAGAAAATCAAGTT CTATGACTTTAGAAAGATCCTGGAGGATAAAAACCTGACAAGCGTGATCATTCCAA AGAGGGGCGGGCGCATCTACATGGCCACCAACCCAGTGACATCCGACTCTACCCCG ATTACATACGCCGGCAAGACTTATAATAGGTGTAACGCTGATGAGGTGGCAGCCGC TAATATCGTTATTTCTGTGCTGGCTCCCCGCAGTAAGAAAAACGAGGAACAGGACG ATATCCCTCTGATTACCAAGAAAGCCGAGAGTAAGTCACCACCGAAAGACCGGAAG AGATCAAAAACAAGCCAGCTGCCTCAGAAA 1202 MASISRPYGTKLRPDARKKEMLDKFFNTLTKGQRVFADLALCIYGSLTLEMAKSLE Parent PESDSELVCAIGWFRLVDKTIWSKDGIKQENLVKQYEAYSGKEASEVVKTYLNSPS Cas12i4 SDKYVWIDCRQKFLRFQRELGTRNLSEDFECMLFEQYIRLTKGEIEGYAAISNMFG NGEKEDRSKKRMYATRMKDWLEANENITWEQYREALKNQLNAKNLEQVVANYKGNA GGADPFFKYSFSKEGMVSKKEHAQQLDKFKTVLKNKARDLNFPNKEKLKQYLEAEI GIPVDANVYSQMFSNGVSEVQPKTTRNMSFSNEKLDLLTELKDLNKGDGFEYAREV LNGFFDSELHTTEDKFNITSRYLGGDKSNRLSKLYKIWKKEGVDCEEGIQQFCEAV KDKMGQIPIRNVLKYLWQFRETVSAEDFEAAAKANHLEEKISRVKAHPIVISNRYW amino acid AFGTSALVGNIMPADKRHQGEYAGQNFKMWLEAELHYDGKKAKHHLPFYNARFFEE sequence VYCYHPSVAEITPFKTKQFGCEIGKDIPDYVSVALKDNPYKKATKRILRAIYNPVA NTTGVDKTTNCSFMIKRENDEYKLVINRKISVDRPKRIEVGRTIMGYDRNQTASDT YWIGRLVPPGTRGAYRIGEWSVQYIKSGPVLSSTQGVNNSTTDQLVYNGMPSSSER FKAWKKARMAFIRKLIRQLNDEGLESKGQDYIPENPSSFDVRGETLYVFNSNYLKA LVSKHRKAKKPVEGILDEIEAWTSKDKDSCSLMRLSSLSDASMQGIASLKSLINSY FNKNGCKTIEDKEKFNPVLYAKLVEVEQRRTNKRSEKVGRIAGSLEQLALLNGVEV VIGEADLGEVEKGKSKKQNSRNMDWCAKQVAQRLEYKLAFHGIGYFGVNPMYTSHQ DPFEHRRVADHIVMRARFEEVNVENIAEWHVRNFSNYLRADSGTGLYYKQATMDFL KHYGLEEHAEGLENKKIKFYDFRKILEDKNLTSVIIPKRGGRIYMATNPVTSDSTP ITYAGKTYNRCNADEVAAANIVISVLAPRSKKNEEQDDIPLITKKAESKSPPKDRK RSKTSQLPQK 1203 MASISRPYGT KLRPDARKKE MLDKFFNTLT KGQRVFADLA LCIYGSLTLE Variant MAKSLEPESD SELVCAIGWF RLVDKTIWSK DGIKQENLVK QYEAYSGKEA Cas12i4 A SEVVKTYLNS PSSDKYVWID CRQKFLRFQR ELGTRNLSED FECMLFEQYI RLTKGEIEGY AAISNMFGNG EKEDRSKKRM YATRMKDWLE ANENITWEQY REALKNQLNA KNLEQVVANY KGNAGGADPF FKYSFSKEGM VSKKEHAQQL DKFKTVLKNK ARDLNFPNKE KLKQYLEAEI GIPVDANVYS QMFSNGVSEV QPKTTRNMSF SNEKLDLLTE LKDLNKGDGF EYAREVLNGF FDSELHTTED KFNITSRYLG GDKSNRLSKL YKIWKKEGVD CEEGIQQFCE AVKDKMGQIP IRNVLKYLWQ FRETVSAEDF EAAAKANHLE EKISRVKAHP IVISNRYWAF GTSALVGNIM PADKRHQGEY AGQNFKMWLE AELHYDGKKA KHHLPFYNAR FFEEVYCYHP SVAEITPFKT KQFGCEIGKD IPDYVSVALK DNPYKKATKR ILRAIYNPVA NTTGVDKTTN CSFMIKREND EYKLVINRKI SRDRPKRIEV GRTIMGYDRN QTASDTYWIG RLVPPGTRGA YRIGEWSVQY IKSGPVLSST QGVNNSTTDQ LVYNGMPSSS ERFKAWKKAR MAFIRKLIRQ LNDEGLESKG QDYIPENPSS FDVRGETLYV FNSNYLKALV SKHRKAKKPV EGILDEIEAW TSKDKDSCSL MRLSSLSDAS MQGIASLKSL INSYFNKNGC KTIEDKEKFN PVLYAKLVEV EQRRTNKRSE KVGRIAGSLE QLALLNGVEV VIGEADLGEV EKGKSKKQNS RNMDWCAKQV AQRLEYKLAF HGIGYFGVNP MYTSHQDPFE HRRVADHIVM RARFEEVNVE NIAEWHVRNF SNYLRADSGT GLYYKQATMD FLKHYGLEEH AEGLENKKIK FYDFRKILED KNLTSVIIPK RGGRIYMATN PVTSDSTPIT YAGKTYNRCN ADEVAAANIV ISVLAPRSKK NREQDDIPLI TKKAESKSPP KDRKRSKTSQ LPQK 1204 MASISRPYGT KLRPDARKKE MLDKFFNTLT KGQRVFADLA LCIYGSLTLE Variant MAKSLEPESD SELVCAIGWF RLVDKTIWSK DGIKQENLVK QYEAYSGKEA Cas12i4 B SEVVKTYLNS PSSDKYVWID CRQKFLRFQR ELGTRNLSED FECMLFEQYI RLTKGEIEGY AAISNMFGNG EKEDRSKKRM YATRMKDWLE ANENITWEQY REALKNQLNA KNLEQVVANY KGNAGGADPF FKYSFSKEGM VSKKEHAQQL DKFKTVLKNK ARDLNFPNKE KLKQYLEAEI GIPVDANVYS QMFSNGVSEV QPKTTRNMSF SNEKLDLLTE LKDLNKGDGF EYAREVLNGF FDSELHTTED KFNITSRYLG GDKSNRLSKL YKIWKKEGVD CEEGIQQFCE AVKDKMGQIP IRNVLKYLWQ FRETVSAEDF EAAAKANHLE EKISRVKAHP IVISNRYWAF GTSALVGNIM PADKRHQGEY AGQNFKMWLR AELHYDGKKA KHHLPFYNAR FFEEVYCYHP SVAEITPFKT KQFGCEIGKD IPDYVSVALK DNPYKKATKR ILRAIYNPVA NTTRVDKTTN CSFMIKREND EYKLVINRKI SRDRPKRIEV GRTIMGYDRN QTASDTYWIG RLVPPGTRGA YRIGEWSVQY IKSGPVLSST QGVNNSTTDQ LVYNGMPSSS ERFKAWKKAR MAFIRKLIRQ LNDEGLESKG QDYIPENPSS FDVRGETLYV FNSNYLKALV SKHRKAKKPV EGILDEIEAW TSKDKDSCSL MRLSSLSDAS MQGIASLKSL INSYFNKNGC KTIEDKEKFN PVLYAKLVEV EQRRTNKRSE KVGRIAGSLE QLALLNGVEV VIGEADLGEV EKGKSKKQNS RNMDWCAKQV AQRLEYKLAF HGIGYFGVNP MYTSHQDPFE HRRVADHIVM RARFEEVNVE NIAEWHVRNF SNYLRADSGT GLYYKQATMD FLKHYGLEEH AEGLENKKIK FYDFRKILED KNLTSVIIPK RGGRIYMATN PVTSDSTPIT YAGKTYNRCN ADEVAAANIV ISVLAPRSKK NREQDDIPLI TKKAESKSPP KDRKRSKTSQ LPQK 1172 GTGCTGCAGCCGCTGCCGCCGATTCCGGATCTCATTGCCACGCGCCCCCGACGACC LDHA GCCCGACGTGCATTCCCGGTACGGTAGGGCCCTGCGCGCACGGCGCCAGAGGGATG GGCGGGTAGAGCCAACTGCCTCTGGTTCTGCTGGCCTCCGCTGCTCGCGAAGGGAT TCCTGCTCCCGGGAGGTGTAGGAGCCGCTTTCCAGAAGCACAGCCCAGAGACGTCT GGGCGGCGGCCCACACAACGCATGTGTTCGGAGCTCGCCGCGCTCTGCTTTTGCTC TAAGCGGGAACCATGGCTTCTGGCCACGCTGGGGAACCGAGGAGGTGGCCGCACCC AAGCAGGGGTCGAAAGCCCGGGTGGATGCGGAACAAGGATATGATAGGCCTTAAGG GTGGGGGATACCTCTGGGCTCGAAATCGGCGGGCGGTGCAAAACTCGAGGTCCAGT TCTCGGAGCCCATAGAGCCAAAAAAGCCTCAGCTTGTCCGGGGCGGGTTCTTGAAA GACGGAAAGCGGCTGAGTACCACGCGGCTTGCATTTTTCTCTTGGGACGCTCGAGA GGTGGGCTCCGTGAGGGCAGCTGCTGCCTGCAGATTATAGGGAGCCCTTTGCGCAT TTATTAAGAAGCTACTGGTGTATCTCGGGCTGCGCTAGGCACGGCGCATGCAAAGA TGAAGCAGGCAGCATCCCAGCCCTTCCGCACCTCAGACGGTCAGTTGAGTAGGATC CGCCGGTACCAACTCCTCCTTTTAACAAATAGGGAGACCGAAAGCTAGGAGACAGT CAGGGATCTCTAAGTTCCCAGTGAGTAGGAGGCAGAGGTGAGGTGTAGAACTCGTT TTTGCATGTCTCTCGCCTCTAGACGCACCCTTCCCTCATCCCATGCCCTCCCACCT CCGCCCCTACATTAAAGGTAGCATTGGATCCCGGGGCCGTTCAGTGAAGCTAGCAG GTGTCCGCAGGAACTCCCTTCCCCCTGCCAGGCTAGAAACCTTACAAGGCTGTCTA GAAATAGCAGTGATTTGTAAGGAGAGACCCGGCTCCAGCTTGGTGACTCTGGGCTG ACTGCCTGCCTAGAGGTCCTCTCGGATTTTTGCCCTTTGGAGTGGTGTCAAAACTA GACGTGATACTTTGGGGATGCAGCCTGTGATATTTCCTCCAGCGAATGCAGTGCAG GGTTGGATTAACAAGGTGGAAAGAATTCGAGGGTTCCACCAAGTAGCTATTAACTC TAGGGCTGCAGGCCTCAGGCCTTCTGCAGCTATTTCTACACTCCCTGTACTGAAAC TATTTCTTCATACTGGGCCTGACAGGCCTTTGCAACAAGGATCACGGCCGAAGCCA CACCGTGCGCCTCCCTCCCGGTTGGTTAACAGGCCCTGGTTTCTAGTATTGCGATT TAAAGTCTGGCGCTGGCTGCGCGCCAGACCTGGGAGGCTGCCAGCTAGGCTTCACG TTGCTGGCGTCTGCTTCGGGGCATTCATTAGGTCTGAAGTCTGAATCCCAGCTCCC TCCCTCTCACCCACTGAGCTGCATAGCTCCAGATTGCCTCTGCTTACGGGCGGGGC TTCTCAGCCTTCTGCCTTCTGGCCCGATGCCCGCTTCCCAACGGCCGGAGGCCGCT AGACTAATCGGCTTCGCCCTGCGCGCTGTAATGCGCATGCGCACGCGCACAAGTTC CTGGGCCCGCCCATCTTCCGGACTTGGGCGGGGCGTAAAAGCCGGGCGTTCGGAGG ACCCAGCAATTAGTCTGATTTCCGCCCACCTTTCCGAGCGGGAAGGAGAGCCACAA AGCGCGCATGCGCGCGGATCACCGCAGGCTCCTGTGCCTTGGGCTTGAGCTTTGTG GCAGTTAATGGCTTTTCTGCACGTATCTCTGGTGTTTACTTGAGAAGCCTGGCTGT GTCCTTGCTGTAGGAGCCGGAGTAGCTCAGAGTGATCTTGTCTGAGGAAAGGCCAG CCCCACTTGGGGTTAATAAACCGCGATGGGTGAACCCTCAGGAGGCTATACTTACA CCCAAACGTCGATATTCCTTTTCCACGCTAAGGTATGGGCCTTCACTCTTCACAGA CCCTGTCATTAGGCCTTTCAACTCTCTTTTGGCAACCATTAGGTTTTTTCCCCTCC CTTTTTAGTCATCTCTAGTGATTTATAGTGGCAAATACCCCCAAAGGAAGTAAAAT AGCTTAAAAAAATCTCTTGGTTAATAAACATTAAAGAAGCTGTAGTGACACTAAAT GTTTTTCCTCCTATAGATTCCTTTTGGTTCCAAGTCCAATATGGCAACTCTAAAGG ATCAGCTGATTTATAATCTTCTAAAGGAAGAACAGACCCCCCAGAATAAGATTACA GTTGTTGGGGTTGGTGCTGTTGGCATGGCCTGTGCCATCAGTATCTTAATGAAGGT AAGTGAGAGTCTACCACACTGGAAGCCCATACCTTGACCCCATCCTCTACCCCCAC TCCTACCCCTAGAACTGTATTATTACATTTCATGTAACAGTATTTAGATTTATGCA CTCATTCGGATAACTTTCTGTGAAACAAACTTTTGAAATATGATAATACACCAAAA GTGTATCTGAAATTAAAAAGAATCAAAGGTTGTCAGGCTGGAGACCCAGTTCCTAA AATTCATTATTCTGTATTAACATGCATGGATTGACTACCAATGAAAAGGAAGGGTC CATGATTTTAAATGAGCCAAAATTCTTTTAAAGTGATTTTTGAATTGAAAATGACA ATTCAAAAATTGTCATTTATTGGTAAAATTATATGGGAAATCATAAGTTCTCCCAC TCAAATCTCATTGCCCCTGTGCCTTGGATAGCAATTTTGTTATCAATTATGGAGCT AAAATTTAATTAGAAAAAAGAAATTGTGAGTAAAGCACTCCTTATTACACTATTGA AAGCTGATTTATATTTAAAAGAAATTGAGGCAGCTTACAACATTAAAATGTCTGAG GCGGGGCACAGTGGCTCATGCTTGTAATGCCAGCACTTTAGGAGGCTGAGGTGGGT GGATCACGAGGTCAGGAGATGGAGACCATCCTGGCTAACACGATGAAACCCCATCT TTACTAGAAATACAAAAAATTAGCCGGGCGTGGTGGCATACGCCTATAGTCCCAGC TACTTGGGAGGCTGAGGCAGGAGAATTGCTTGAACCCAGGAGGTGGAGGTGGCAGT GACCCGAGATAGCACCACTCCACTCCAGCCTGGGCGACAGTGAGACTCCATCTCAA AAAAAAAAATCTGAAGTTAAGATGTGGAGTGTCTAATAAAAGTAAAATGATGAATT CTGGGTTCTAAATAGAAATGGATTCAAGTGAGAAGGGACTAAAGACAGAAATGAGC TATGAAAAGGCCTCGTAACAACACAGGTGACTCTACATATGTTCTTAGGAAAGGCC ACATAATACACCAACTTTTATTCCTTACCCACTAGATGAGAAATTGATGCTGTTTT CCCCACACCTACAAACCGCCTATGTTTTTTCTCTGTGATGGCCTCTGGCTCAGGTG TGGGTAAGAAGAGTAACTGACACTCATTATATTGTGGATGATTTAGGGATAGATCT GCAGCTTGAATAACTTTTGGTAACGATAGACCACATCCAGTTGTATTAAAGCTGTT ATTGGTGCTCCTGGCCTGAAATGGACCTATGAACTTTGAGTTGCAACTATAAGGAT ATTTTTTGCCAGTATTATACACTGCACAAACCTATTTATCCATAACTGTTAGTATT GGTTCATATATGGAATCAACCAGGGAATAGTTCAGATTCCATCTCTGAAAGATGGG CGGAAATCAGACTTTTTAACTTTTTAAGTTTTTTTTTTTTGAGACGGAATCTCGCT TTGTTGCCCTGGCTGGAGTGCAGTGGCACGATCTTGGCTCACTTGACCTCCTGGGT TCAAGTGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCACCCACCG CCACGCCTGGCTGATTTTTGTATTTTTAGTAGAGACAGGCCTTCACCATATTGGCC AGGCTGGTCTTTTTTTTTTTTTTTTTTTTTTTTTTCTGAGAAGGAGTCTCGCCGTG TCGCCCAGGCTAGAGTGCAGTGGCGTGAACTCCGCTCACTGCTAGCTCTGCCTCCC GGGTTCATACCATTCTCCTGTCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACCC ACCACCACGCCTGGCTAAATGTTTGTATTTTTTAGTAGAGACGGGGTTTCACCATG TTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCGCCTACCTTGGCCTCCCA AAGTGCTGGGATTACAAGCGTGAGCCACCGTGCCTGGCCTGGCCAGGCTGTCTTGA ACTCCTGACCTCAAGTGATGTGCCCGCCTCGGCCTCCCAAAGTGTTGGGATTACAG ATGTGAGTCACTATGCCCGGCCAGAACATTTCTTACTAATTTCAAGTCTTGATGCT GGTCAATATCACCTAGTTAAATGAATAACAACCTAAAATTGGTGTGTAGGATGGAA TTTGAGAGAGTAGACAGAGCAGTTTTATATAATTGGAAGTTATTCTAGCAACTGCC AGTCCAGTGTTCTGCTTCCACATCTGCAGTGGTGGAACTCCTATAGAGCTCGCTTC AGTGGGGAGACAGGGCTGGAGAGAGGGTCAGTGCTATCTATGTAGGGTGTAATCTG TAAGTCAGCTTTTGAAATGGGGTGCCCTCTACTTTGAATATCTCGATACTGTACTA ATAAAGTAACAGAACTCTCCTATGCCAGAAATATAGAAATTTTTCATGCTCTTCTA AAAATCTAGAAGTGGCAATTTTCCATTTAACTAAAGATTTGATGTCTTTTAGGACT TGGCAGATGAACTTGCTCTTGTTGATGTCATCGAAGACAAATTGAAGGGAGAGATG ATGGATCTCCAACATGGCAGCCTTTTCCTTAGAACACCAAAGATTGTCTCTGGCAA AGGTTGATTTCAACAAGTTTATATTATAATCCATGCTTGACTTAAATTCTTTTTCC AGATGGTCTCCATTTGTTGCTTAGGGTAGAGTGCAGTTGCACAATTATGGCTCACC ACAGCCTCGAACCCTGGGCTCAAGCAATCCTCCTTCCACTTCATTACCCCCTCCCC CTCACAAAGAAACTGGGACTATAGGGTATGCTACCATGCCCGGCTAATTTTTTTAC TTTTTGTAGAGATGGGGACCCACTGTGTTGCCCAGGCCTGTCTTGAACCACTGGGC TCAAGTGATCCTCCCTCCTTAGCCTTCCGAAGTACTGGGATTGCAGGTGTGAACCA CTGTGCCCGGCTTTAGACTTAAATGTTTTATCAGGCTTGAAATCCTAGCTCTTTAA AGATTTTGTTTTAAATGCCGGGTGCAAGAGCCTGGGAACAATTTCACTTAGGTGCC TGTGAATATCAAAGTTTCAATTTCTGGCAAATGGTTTAAAATAGAAATCCAATTTG TCCATGCTATGCAAACCATCTGAATTAGAATGTAATGAGTAAAGCTTAAACCTTAG GTCTGTATTTAACCACATTGTGTTACTTACTTGCCCCCACATCCTTTCACACACGA AGTTGAGAATAGGGTAAATAAATGAGCCTGTTCAGCTAATACTCTTGGCTTGACCC TTTCACACTTAACAGCACCAGCCAAGAAACCTGAATGTGAGCCCAAATAGTGTCTA TTTTGATACCTGAAAATCACTGGCCACCTTGCTGATGGGCAACTCCCTTCATCACT GGTTTAACTCTCTTGTGCCATAGGGTATCTAGAAGCAAAATATGTTTGTTAAGTGT AAAGCTGTCTCTGCTTAAAAACAAGTCCCCCTACCACCACCACCACACACACACAC ACACACACACACACACACACACACACACACACACACACACGAAATTGCCTGTTCCT GGGCTGATAGGACACCAGTTAAGTAGAAACAGGAGTATGGAAGAGTGTGAACGTTG AGCTTGGGGATCAAAAATTTGAGGATATGTAAGAAATTAATAGGAGAATCAAATAA TAAACTTGATTTCCTCCAGCTCTCCCTAATTGTAGTTACATAAAGTTACAACTTGA CTAAAACTACAAGGAAGATGTTGACATGCTCTTCCTCCATTTAAGAAGCCATAATG ATAAAACTCTAAGAACAAGAAAGGTTTGTGGAGCATTTATGGAACAAATTTTTGCT GCCTAGGTAAAATTTATTCTAAAGGCCTTAATCTGGTCATTATTCCCCTTTTCTCT AGACTATAATGTAACTGCAAACTCCAAGCTGGTCATTATCACGGCTGGGGCACGTC AGCAAGAGGGAGAAAGCCGTCTTAATTTGGTCCAGCGTAACGTGAACATCTTTAAA TTCATCATTCCTAATGTTGTAAAATACAGCCCGAACTGCAAGTTGCTTATTGTTTC AAATCCAGGTGAGGCTTTTGACTGCATAAAAATTGACAAGCTATAGTAAAACTGAT AGTATATGATATATATATTATATATATTTTAAATATTTTGAAATATTTTAAAAAAT ACATTTTTAAAAATATTTTCGAATATTATTTTAAAATATATATATATATTTTGAGG CGGAGTTTTGCTCTTGTCGCCCAGGTTGGAGTGCAGTGGCGCAATCTGGGCTCACT GCAACCTCTGCCTCATGGGTTCAAGCGATTCTTTTGCCTCAGCCTCTCAAGTAGCT GGGATTATAAGCGCCTGCCACCACACATGGCTAATTTTTTATATTTTTAGTAGAGA CAGGGTTTCACCATGTTGGCCAGGCTGGTTTTGAACTCCTGGCCTCAAGCAGTCCA TCTGCCTCCCAAAGTGCTAGGATTACAGGCGTGAGCCACCGTGCCCAGCCACGCAT ATTTATTGATTCATTTATTTTTCTTTTTTTTTTTTTTTTTTTTTTGAGACGGAGTC TTGCTCTGTCACCCTGGCTGGAGTACAGTGGCTTGATCTTGGCTCACTGCAAGCTC CGCCTCCCGGGTTCATGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTAC AGGTGCCCACCACGACGCCTGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTT CATCAGGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCTGCCCGCCTTGG CCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGTGCCTGGTGATTCATTTAT TTTTCATGTTTCATTTCCCTTCTAAGGAGATTTGTGTGTGTGTGTTTTTTGTTTTT TAATAATTTTAAAACATTAAAGGGAATACAATGCCTTTAAATGTAGTTGGAGCTTA AAATTACCTGCCCAAGATCTTGGATAAGGGATAAGTTTGTGAATAATTGTTATTCT CTTTTTTTTTTTTTTTTTTTTTGAGACAGTCTCACTTTGTAGCTCAGGCTGGAGTG CAGTGGTTCGATCTTGGCTCACTGCAACCTCTGCCTCCTGGGTTCAAGCAATTCTC CTGCCTCAGCCTCCCAAGAGCTGGGATTACAGGCACGTGCCACCATGCTCGGCTAA TTTTTGAAGTTTTAGTAGAAAGGGGTTTCACCATGTTGCCCAGGCTGGTCTCAAAT TCCTGAGCTCAGGTGATCCATCTGCCTCAGCCTCCCAAAGTATTAGGATTACAGGC GTGAGCCACCGTGCCCGGGCCCATAATTGTCTCTTAGTTGATAAACAGTTTATTTT CATAAAACTGTTACTATACTTTTTTTTTGAGAGCATGTCTCACTCTGTCGCCCAAG CTGGAGGGCAATGGGATGATCATGGCAGCTTTGACCTACTAGGCTCAGGTGATCCT TCTTCCTCAGCCTCTTAAGTAGCTAGGACTACAGGCGTGCACCAATATGCCTGGCT AGTTTGTTAAAAGTTTTTTTGTAGAGATGGGGTTTTGCTATGTTGCCCAGGCTGGT CTTGAACTGCTGGCCTCAGGCAGTCCTCCCACCTCAGCCTCCCAAAGTGTTGGGAT AACAGGTGTGAGTTGTCATGCCCAGCCAAAACTACTTTTTGAATAATTAATGGACT TGATATACATAGTGTAGAGGCTTAAAAATATTAACAAAATTATTGGTTAGCCATGA TCAATATCAAGATCCTGAAAAGCCATATATCTGGAGTAGCCTATTATTATCTAATG ATCACCTAGTATCTGGTTAAGTGTTTTCTTCATAGTAGGTATATCTTTTTTGTGTG TAGGGAGAGGATAATGGGTGATTTTTATTTTCTCCTTTTTCATAGTGGATATCTTG ACCTACGTGGCTTGGAAGATAAGTGGTTTTCCCAAAAACCGTGTTATTGGAAGCGG TTGCAATCTGGATTCAGCCCGATTCCGTTACCTAATGGGGGAAAGGCTGGGAGTTC ACCCATTAAGCTGTCATGGGTGGGTCCTTGGGGAACATGGAGATTCCAGTGGTAAG CATAAGTTATTTTCTTTTTGTTTTTGAAAAGATTATATAAAAAGTCGATGGGCATT ATATTATTCAATTAGAGCCTAATCAAATATCCATTCAGTAGGATGGAATGGTTTCC CGAAATCTAGCATTTTGTATAATTATATGTTAAGAATTGTTAAGATTGTTGCCATT TTATATGGCATTTTATGGCGAGGGGGACGGGAAATGAAATTTCTCTTCTTACCATG GATATCTTAAGACTGTAGTTCTTAGGATGTCTTCAGTCATTTAATATCACAGCTGT TTATACCTGACTTGTACTGCCTGGCCCTGAAAAGATGAGCAAATCCAAATGCACAA AAGTTATATTATCACAGTTGAAAAATGTTATGATTAGGTTCTGTATGCTAAGAAAA CCCCCCTTATGTTCTCATACTATCTTTATATTTCAAATATACATGGGTTAAACATT TCAATTGGCTAGAGAAACAGGTTAGAATACAGTTAAAATTCTTAGTTTTACATAAT GTAAGTAAATGAAAATCTAATCTAAAAGTGAGTAATGACTACATTAGTAGTCTTGA CCATCTACCAAAATTGAGTATTCTTCCTCCGAAGATAAGAGAATTAGGAAAATGAA TCACAATTACTAATCTGTTGGTACATGAAAATAAATGTAGTCTGTACTATTTCTTT TAGTGCCTGTATGGAGTGGAATGAATGTTGCTGGTGTCTCTCTGAAGACTCTGCAC CCAGATTTAGGGACTGATAAAGATAAGGAACAGTGGAAAGAGGTTCACAAGCAGGT GGTTGAGAGGTAATAAATCTTTCAATTTGGCAACACAGAATATTAACATTTACTAT TTTTATTTAAAAGGTTAAAATTGTAATAGTATTTGCATTTGAGAACTTTTTGTTAG AAAACTTGTGTGGTTTTTTTGTTTTGTTTTGTTTGAGACAGAATCTTGCCCTTTCG CACAGGCTGCAGTGCAGTGGCGCAATCTTGGCTCACTGCAACCTCTGCCTCCCGGG TTCAGGCGATTCTCCTGCCTCAGCCTCCTGGGTACCTGGGACTACAGGCATATGCC ATGACGCCCGGCTAATTTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTAG CCAAAAAAAAAAGAATGTGCCTCACCTTGCAAGGCCCAGGCCCTAGGATCACTTGA GCTCAGGAGTTCAAGGCCAGCCTGGGCAACAGGGCAAAACCCTGTCTCTACAATAA ATACACAAATTAGCCAGGCATGGTGGTGAGCACCTGTGGTCCTAGCTACTTGAGAG GCTGAGGCAGGAGGATCGCATGAGCCTGGGAGGTCAAGGCTGCAGTGAAGCGAGAT CCTGCCACTGCACTCCAGAGCCTGCTAGCCTGGGTGACAGAGTAAGAGCCTGTCTC AAAGGAAAAAAAAAATTATTGAAATAGGGAAGCTTTCAACTTGGTGGCATTATTTA CCTTTGTGGTCCTGTGTGGACCTCAGGTCTATAGAATTAAAAAATGAATCATAGCC GGGCATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCAGA TCACGAGGTCAGGAGATGGAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTA CTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCAGGCGCCTGTAGTCCCAGCTAC TCGGGAGGCTGAGGCAGGAGAATGGCATGAACCCGGTAGTTGGAGCTTGCAGTGAG CCGAGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCAAA AAAAAAAGAATCATAATCTTTAGTTCATAACATATTCTTGTGATTGGTCAAGCAAG GCCCTCTTGTTTGTATTTGTTTAATTAAATAAAACCTGTGAACCCACCACCCAGCT CAAGAAAGAAACACAATATCTGTCAAATAACATTGTTGAATCAGAATTTAGTATTC TGCTGGTGTTTGGAAATAAGTGGATTCTGTGCTCTTTCCCCCAGCTATCCCTCTGT CCCCCTCACGCTCCCACTTGAGATAATCCTGAGTTAAGGATGCTATGTTATCTTGG ATTTCTTTTTAAAATTCAATATTATATTTTTAAGAATTATCCAATTTTTTTTACAA GTAGCTATAGTTTATTTTTTGATAGCTGTGTAATATTCCATTGTATCAGTATACCA TGATTTATCCATTCTTCTGTTGGAGGACATTGGAAAGATTGTCATGTTTTTGCTGT TACTAACAGTACTGTTAATGAATATCCCTGTACATAATATCCTAGCATACATGTGT GCAAGGGTTATTCTTGGTATAATGCAACATTGTGGCATTATTTACTGTAAAATGTG TATTAATGAAAACTTTGTTTTTCTTTCTTTCTCCCACCCTGCTTTTTCTGCCTTTA CCTATGGTTTCCTATCATACAGTGCTTATGAGGTGATCAAACTCAAAGGCTACACA TCCTGGGCTATTGGACTCTCTGTAGCAGATTTGGCAGAGAGTATAATGAAGAATCT TAGGCGGGTGCACCCAGTTTCCACCATGATTAAGGTAGGTCTATGTAGTGATACGC TGCATTTGAATGCTTTTTGCTGGCTTTTTAAAAAAGATTCTTCTGAGAAAGATTAA TACAAGTCTTCCATTACTGACTTAAGTGAAATAAATTAATGTACCCACAGCTTACC TTTTTTGAAAGAAATGGTTGAGCTTTAGGATTAATGTCCATTAGGCCTGTTCAACA CATAGATACTTGATAATTTGACTACAAAAAAGTCTTGTTCAATTATGCTGAGGTAG GTGGAAGACTATAAAAGAAATAAACTATTTCTCCATTGGGGAAAATAGAAATTATA TTCAAGTTAGCATTATGTTACTATTTTTAATGACTTTCTTTTATACTATTAATTAA ATCATAACTGAACACCTGGAAAGGAATTTCTACTTATCAAAGTTTTTTATTTTTTT GAGACAGTCTCCCTCTGTCACCCAGGCTGCAGTGCAGTGGCCGATCTCGGCTCACC GCAACCTCTGCCTCCCAGGTTTAAGCGATTCTTCTGCCTCAGCCTCCTAAGTAGCT GGGACTACAGGTGCGTGCCACCACGCCCGGCTACTTTTTGTATTTTTAGTAGAGAT GGAGTTTCACCATATTGGCTAGGCTGGTCTCGAACTCCTGACCTTGTGATCCACCC GCCTCGGCCTCCCCGAATGCTGGGATTGCAGGTGTGAGCCACCGCACCTGGCCTCA AGTTGTATTTTAAAATCTTCATAATTAGGCCACACACAGTGACTGACAGCTGTAAT GCCAGCACTTTGGAAGGCCAAGGGCAGGAGAATTGCTTGAGCCCAGGTGTTTGAGA CCACCCTAGGCAGTATAGTGAGATCTTGCCTCTGTTAAAAAAAAAAAAAAAAAAAA AGGCCATGTGCGGGCAGCTGATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGGG TGGATCACCTGAGGTCAGTAGTTCAAGACCAGCCTGACCAACATGGTGAAACCCTG TCTCTACTAAAAATACAGAATTAGCCAGGTGTGGTGGCAGGCGCCTGTAATCCCAG CTACTTGGGAGACTGAGGCAGAAGAATCACTTGAACCCAGGAGGTGGAGGTTGCAG TGAGCTGAGATCGCACCATTGCACTCCAGCCTGGGCAACAAGAGTGAAACTCCATC TCAAAAGAAAAAAAAAAGCGGCTGGGCTCTGTGGCTCATGCTTGTAACCCCAGCAC TTTGGGAGGCCAAGAGGTGGATCACCTGAGGTCAAGAATTTGAGACCAACCTGGCC AACATGGTGAAACCCCATCTGTACTAAACATACAAAAATTAGCCAAGTGTGGTGGC GCACGCCTGTAGTCCCAGAAGGCTGAAGCAGGAGAATTACTTGAACCCTGGAGGTG GAGGTTGCGGTGAGCTGAGATCGTGCCACTGCACTCCAGCCTGGGCGACAGAGCGA GACTCTGCCTCAAAAAAAAATTAAAAAAAAAAAGCTTTATAATTATAGAGACTGTA AGTCTTGGGAAACCTGGGAATGCATAGACAAAATGTGAGATTTTTTTTTTTTCATT TCATCTTCAGGGTCTTTACGGAATAAAGGATGATGTCTTCCTTAGTGTTCCTTGCA TTTTGGGACAGAATGGAATCTCAGACCTTGTGAAGGTGACTCTGACTTCTGAGGAA GAGGCCCGTTTGAAGAAGAGTGCAGATACACTTTGGGGGATCCAAAAGGAGCTGCA ATTTTAAAGTCTTCTGATGTCATATCATTTCACTGTCTAGGCTACAACAGGATTCT AGGTGGAGGTTGTGCATGTTGTCCTTTTTATCTGATCTGTGATTAAAGCAGTAATA TTTTAAGATGGACTGGGAAAAACATCAACTCCTGAAGTTAGAAATAAGAATGGTTT GTAAAATCCACAGCTATATCCTGATGCTGGATGGTATTAATCTTGTGTAGTCTTCA ACTGGTTAGTGTGAAATAGTTCTGCCACCTCTGACGCACCACTGCCAATGCTGTAC GTACTGCATTTGCCCCTTGAGCCAGGTGGATGTTTACCGTGTGTTATATAACTTCC TGGCTCCTTCACTGAACATGCCTAGTCCAACATTTTTTCCCAGTGAGTCACATCCT GGGATCCAGTGTATAAATCCAATATCATGTCTTGTGCATAATTCTTCCAAAGGATC TTATTTTGTGAACTATATCAGTAGTGTACATTACCATATAATGTAAAAAGATCTAC ATACAAACAATGCAACCAACTATCCAAGTGTTATACCAACTAAAACCCCCAATAAA CCTTGAACAGTGACTACTTTGGTTAATTCATTATATTAAGATATAAAGTCATAAAG CTGCTAGTTATTATATTAATTTGGAAATATTAGGCTATTCTTGGGCAACCCTGCAA CGATTTTTTCTAACAGGGATATTATTGACTAATAGCAGAGGATGTAATAGTCAACT GAGTTGTATTGGTACCACTTCCATTGTAAGTCCCAAAGTATTATATATTTGATAAT AATGCTAATCATAATTGGAAAGTAACATTCTATATGTAAATGTAAAATTTATTTGC CAACTGAATATAGGCAATGATAGTGTGTCACTATAGGGAACACAGATTTTTGAGAT CTTGTCCTCTGGAAGCTGGTAACAATTAAAAACAATCTTAAGGCAGGGTGCAGTGG CTCATGCCTATAATCCCAGCACTTTGGGAAGCCCAGGTGGGCTGATCACTGGAGGC CAGGAATTGGGGACCAGCCTGGCCAACACAACAAAACCCCATCTGTTAAAAAAACA AAACAAAACCAAAAAAAACAAGTAACCTTGGTGGATGTCTACTCAAGTTTTCTGCA CATTTTTCTGAAAATACAACTGTGACCCTTA 1173 GTGCTGCAGCCGCTGCCGCCGATTCCGGATCTCATTGCCACGCGCCCCCGACGACC LDHA GCCCGACGTGCATTCCCGGTACGGTAGGGCCCTGCGCGCACGGCGCCAGAGGGATG exon 1 GGGGGGTAGAGC 1174 CCAGCAATTAGTCTGATTTCCGCCCACCTTTCCGAGCGGGAAGGAGAGCCACAAAG LDHA CGCGCATGCGCGCGGATCACCGCAGGCTCCTGTGCCTTGGGCTTGAGCTTTGTGGC exon 2 AGTTAATGGCTTTTCTGCACGTATCTCTGGTGTTTACTTGAGAAGCCTGGCTGTGT CCTTGCTGTAGGAGCCGGAGTAGCTCAGAGTGATCTTGTCTGAGGAAAGGCCAGCC CCACTTGGGGTTAATAAACCGCGATGGGTGAACCCTCAGGAGGCTATACTTACACC CAAACGTCGATATTCCTTTTCCACGCTAAGGTATGGGCCTTCACTCTTCACAGACC CTGTCATTAGGCCT 1175 AATAAACATTAAAGAAGCTGTAGTGACACTAAATGTTTTTCCTCCTATAGATTCCT LDHA TTTGGTTCCAAGTCCAATATGGCAACTCTAAAGGATCAGCTGATTTATAATCTTCT exon 3 AAAGGAAGAACAGACCCCCCAGAATAAGATTACAGTTGTTGGGGTTGGTGCTGTTG GCATGGCCTGTGCCATCAGTATCTTAATGAAGGTAAGTGAGAGTCTACCACACTGG AAGCCCATACCTTGACCCCATCCTCT 1176 AATCTAGAAGTGGCAATTTTCCATTTAACTAAAGATTTGATGTCTTTTAGGACTTG LDHA GCAGATGAACTTGCTCTTGTTGATGTCATCGAAGACAAATTGAAGGGAGAGATGAT exon 4 GGATCTCCAACATGGCAGCCTTTTCCTTAGAACACCAAAGATTGTCTCTGGCAAAG GTTGATTTCAACAAGTTTATATTATAATCCATGCTTGACTTAAATTCTTT 1177 AAAATTTATTCTAAAGGCCTTAATCTGGTCATTATTCCCCTTTTCTCTAGACTATA LDHA ATGTAACTGCAAACTCCAAGCTGGTCATTATCACGGCTGGGGCACGTCAGCAAGAG exon 5 GGAGAAAGCCGTCTTAATTTGGTCCAGCGTAACGTGAACATCTTTAAATTCATCAT TCCTAATGTTGTAAAATACAGCCCGAACTGCAAGTTGCTTATTGTTTCAAATCCAG GTGAGGCTTTTGACTGCATAAAAATTGACAAGCTATAGTAAAACTGATAG 1178 GTGTGTAGGGAGAGGATAATGGGTGATTTTTATTTTCTCCTTTTTCATAGTGGATA LDHA TCTTGACCTACGTGGCTTGGAAGATAAGTGGTTTTCCCAAAAACCGTGTTATTGGA exon 6 AGCGGTTGCAATCTGGATTCAGCCCGATTCCGTTACCTAATGGGGGAAAGGCTGGG AGTTCACCCATTAAGCTGTCATGGGTGGGTCCTTGGGGAACATGGAGATTCCAGTG GTAAGCATAAGTTATTTTCTTTTTGTTTTTGAAAAGATTATATAAAAAGT 1179 CTAATCTGTTGGTACATGAAAATAAATGTAGTCTGTACTATTTCTTTTAGTGCCTG LDHA TATGGAGTGGAATGAATGTTGCTGGTGTCTCTCTGAAGACTCTGCACCCAGATTTA exon 7 GGGACTGATAAAGATAAGGAACAGTGGAAAGAGGTTCACAAGCAGGTGGTTGAGAG GTAATAAATCTTTCAATTTGGCAACACAGAATATTAACATTTACTATTTT 1180 TTCTCCCACCCTGCTTTTTCTGCCTTTACCTATGGTTTCCTATCATACAGTGCTTA LDHA TGAGGTGATCAAACTCAAAGGCTACACATCCTGGGCTATTGGACTCTCTGTAGCAG exon 8 ATTTGGCAGAGAGTATAATGAAGAATCTTAGGCGGGTGCACCCAGTTTCCACCATG ATTAAGGTAGGTCTATGTAGTGATACGCTGCATTTGAATGCTTTTTGCTGGCTTTT 1181 GGAATGCATAGACAAAATGTGAGATTTTTTTTTTTTCATTTCATCTTCAGGGTCTT LDHA TACGGAATAAAGGATGATGTCTTCCTTAGTGTTCCTTGCATTTTGGGACAGAATGG exon 9 AATCTCAGACCTTGTGAAGGTGACTCTGACTTCTGAGGAAGAGGCCCGTTTGAAGA AGAGTGCAGATACACTTTGGGGGATCCAAAAGGAGCTGCAATTTTAAAGTCTTCTG ATGTCATATCATTTCACTGTCTAGGCTACAACAGGATTCTAGGTGGAGGTTGTGCA TGTTGTCCTTTTTATCTGATCTGTGATTAAAGCAGTAATATTTTAAGATGGACTGG GAAAAACATCAACTCCTGAAGTTAGAAATAAGAATGGTTTGTAAAATCCACAGCTA TATCCTGATGCTGGATGGTATTAATCTTGTGTAGTCTTCAACTGGTTAGTGTGAAA TAGTTCTGCCACCTCTGACGCACCACTGCCAATGCTGTACGTACTGCATTTGCCCC TTGAGCCAGGTGGATGTTTACCGTGTGTTATATAACTTCCTGGCTCCTTCACTGAA CATGCCTAGTCCAACATTTTTTCCCAGTGAGTCACATCCTGGGATCCAGTGTATAA ATCCAATATCATGTCTTGTGCATAATTCTTCCAAAGGATCTTATTTTGTGAACTAT ATCAGTAGTGTACATTACCATATAATGTAAAAAGATCTACATACAAACAATGCAAC CAACTATCCAAGTGTTATACCAACTAAAACCCCCAATAAACCTTGAACAGTGACTA CTTTGGTTAATTCATTATATTAAGATATAAAGTCATAAAGCTGCTAGTTATTATAT TAATTTGGAAATATTAGGCTATTCTTGGGCAACCCTGCAACGATTTTTTCTAACAG GGATATTATTGACTAATAGCAGAGGATGTAATAGTCAACTGAGTTGTATTGGTACC ACTTCCATTGTAAGTCCCAAAGTATTATATATTTGATAATAATGCTAATCATAATT GGAAAGTAACATTCTATATGTAAATGTAAAATTTATTTGCCAACTGAATATAGGCA ATGATAGTGTGTCACTATAGGGAACACAGATTTTTGAGATCTTGTCCTCTGGAAGC TGGTAACAATTAAAAACAATCTTAAGGCAGGGTGCAGTGGCTCATGCCTATAATCC CAGCACTTTGGGAAGCCCAGGTGGGCTGATCACTGGAGGCCAGGAATTGGGGACCA GCCTGGCCAACACAACAAAACCCCATCTGTTAAAAAAACAAAACAAAACCAAAAAA AACAAGTAACCTTGGTGGATGTCTACTCAAGTTTTCTGCACATTTTTCTGAAAATA CAACTGTGACCCTTA 1211 MSNKEKNASETRKAYTTKMIPRSHDRMKLLGNFMDYLMDGTPIFFELWNQFGGGID (Cas12i1 of RDIISGTANKDKISDDLLLAVNWFKVMPINSKPQGVSPSNLANLFQQYSGSEPDIQ SEQ ID AQEYFASNFDTEKHQWKDMRVEYERLLAELQLSRSDMHHDLKLMYKEKCIGLSLST NO: 3 of AHYITSVMFGTGAKNNRQTKHQFYSKVIQLLEESTQINSVEQLASIILKAGDCDSY U.S. Pat. RKLRIRCSRKGATPSILKIVQDYELGTNHDDEVNVPSLIANLKEKLGRFEYECEWK No. CMEKIKAFLASKVGPYYLGSYSAMLENALSPIKGMTTKNCKFVLKQIDAKNDIKYE 10,808,245) NEPFGKIVEGFFDSPYFESDTNVKWVLHPHHIGESNIKTLWEDLNAIHSKYEEDIA SLSEDKKEKRIKVYQGDVCQTINTYCEEVGKEAKTPLVQLLRYLYSRKDDIAVDKI IDGITFLSKKHKVEKQKINPVIQKYPSFNFGNNSKLLGKIISPKDKLKHNLKCNRN QVDNYIWIEIKVLNTKTMRWEKHHYALSSTRFLEEVYYPATSENPPDALAARFRTK TNGYEGKPALSAEQIEQIRSAPVGLRKVKKRQMRLEAARQQNLLPRYTWGKDFNIN ICKRGNNFEVTLATKVKKKKEKNYKVVLGYDANIVRKNTYAAIEAHANGDGVIDYN DLPVKPIESGFVTVESQVRDKSYDQLSYNGVKLLYCKPHVESRRSFLEKYRNGTMK DNRGNNIQIDFMKDFEAIADDETSLYYFNMKYCKLLQSSIRNHSSQAKEYREEIFE LLRDGKLSVLKLSSLSNLSFVMFKVAKSLIGTYFGHLLKKPKNSKSDVKAPPITDE DKQKADPEMFALRLALEEKRLNKVKSKKEVIANKIVAKALELRDKYGPVLIKGENI SDTTKKGKKSSTNSFLMDWLARGVANKVKEMVMMHQGLEFVEVNPNFTSHQDPFVH KNPENTFRARYSRCTPSELTEKNRKEILSFLSDKPSKRPTNAYYNEGAMAFLATYG LKKNDVLGVSLEKFKQIMANILHQRSEDQLLFPSRGGMFYLATYKLDADATSVNWN GKQFWVCNADLVAAYNVGLVDIQKDFKKK 1212 MSISNNNILPYNPKLLPDDRKHKMLVDTFNQLDLIRNNLHDMIIALYGALKYDNIK (Cas12i3 of QFASKEKPHISADALCSINWFRLVKTNERKPAIESNQIISKFIQYSGHTPDKYALS SEQ ID HITGNHEPSHKWIDCREYAINYARIMHLSFSQFQDLATACLNCKILILNGTLTSSW NO: 14 of AWGANSALFGGSDKENFSVKAKILNSFIENLKDEMNTTKFQVVEKVCQQIGSSDAA U.S. Pat. DLFDLYRSTVKDGNRGPATGRNPKVMNLFSQDGEISSEQREDFIESFQKVMQEKNS No. KQIIPHLDKLKYHLVKQSGLYDIYSWAAAIKNANSTIVASNSSNLNTILNKTEKQQ 10,808,245) TFEELRKDEKIVACSKILLSVNDTLPEDLHYNPSTSNLGKNLDVFFDLLNENSVHT IENKEEKNKIVKECVNQYMEECKGLNKPPMPVLLTFISDYAHKHQAQDFLSAAKMN FIDLKIKSIKVVPTVHGSSPYTWISNLSKKNKDGKMIRTPNSSLIGWIIPPEEIHD QKFAGQNPIIWAVLRVYCNNKWEMHHFPFSDSRFFTEVYAYKPNLPYLPGGENRSK RFGYRHSTNLSNESRQILLDKSKYAKANKSVLRCMENMTHNVVFDPKTSLNIRIKT DKNNSPVLDDKGRITFVMQINHRILEKYNNTKIEIGDRILAYDQNQSENHTYAILQ RTEEGSHAHQFNGWYVRVLETGKVTSIVQGLSGPIDQLNYDGMPVTSHKFNCWQAD RSAFVSQFASLKISETETFDEAYQAINAQGAYTWNLFYLRILRKALRVCHMENINQ FREEILAISKNRLSPMSLGSLSQNSLKMIRAFKSIINCYMSRMSFVDELQKKEGDL ELHTIMRLTDNKLNDKRVEKINRASSFLTNKAHSMGCKMIVGESDLPVADSKTSKK QNVDRMDWCARALSHKVEYACKLMGLAYRGIPAYMSSHQDPLVHLVESKRSVLRPR FVVADKSDVKQHHLDNLRRMLNSKTKVGTAVYYREAVELMCEELGIHKTDMAKGKV SLSDFVDKFIGEKAIFPQRGGRFYMSTKRLTTGAKLICYSGSDVWLSDADEIAAIN IGMFVVCDQTGAFKKKKKEKLDDEECDILPFRPM 1230 ATGGCAACTCTAAAGGATCAGCTGATTTATAATCTTCTAAAGGAAGAACAGACCCC LDHA CCAGAATAAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATGGCCTGTGCCATCA isoform 1 GTATCTTAATGAAGGACTTGGCAGATGAACTTGCTCTTGTTGATGTCATCGAAGAC cDNA AAATTGAAGGGAGAGATGATGGATCTCCAACATGGCAGCCTTTTCCTTAGAACACC AAAGATTGTCTCTGGCAAAGACTATAATGTAACTGCAAACTCCAAGCTGGTCATTA TCACGGCTGGGGCACGTCAGCAAGAGGGAGAAAGCCGTCTTAATTTGGTCCAGCGT AACGTGAACATCTTTAAATTCATCATTCCTAATGTTGTAAAATACAGCCCGAACTG CAAGTTGCTTATTGTTTCAAATCCAGTGGATATCTTGACCTACGTGGCTTGGAAGA TAAGTGGTTTTCCCAAAAACCGTGTTATTGGAAGCGGTTGCAATCTGGATTCAGCC CGATTCCGTTACCTAATGGGGGAAAGGCTGGGAGTTCACCCATTAAGCTGTCATGG GTGGGTCCTTGGGGAACATGGAGATTCCAGTGTGCCTGTATGGAGTGGAATGAATG TTGCTGGTGTCTCTCTGAAGACTCTGCACCCAGATTTAGGGACTGATAAAGATAAG GAACAGTGGAAAGAGGTTCACAAGCAGGTGGTTGAGAGTGCTTATGAGGTGATCAA ACTCAAAGGCTACACATCCTGGGCTATTGGACTCTCTGTAGCAGATTTGGCAGAGA GTATAATGAAGAATCTTAGGCGGGTGCACCCAGTTTCCACCATGATTAAGGGTCTT TACGGAATAAAGGATGATGTCTTCCTTAGTGTTCCTTGCATTTTGGGACAGAATGG AATCTCAGACCTTGTGAAGGTGACTCTGACTTCTGAGGAAGAGGCCCGTTTGAAGA AGAGTGCAGATACACTTTGGGGGATCCAAAAGGAGCTGCAATTTTAA 1231 ATGGCAACTCTAAAGGATCAGCTGATTTATAATCTTCTAAAGGAAGAACAGACCCC LDHA CCAGAATAAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATGGCCTGTGCCATCA isoform 2 GTATCTTAATGAAGGACTTGGCAGATGAACTTGCTCTTGTTGATGTCATCGAAGAC cDNA AAATTGAAGGGAGAGATGATGGATCTCCAACATGGCAGCCTTTTCCTTAGAACACC AAAGATTGTCTCTGGCAAAGTGGATATCTTGACCTACGTGGCTTGGAAGATAAGTG GTTTTCCCAAAAACCGTGTTATTGGAAGCGGTTGCAATCTGGATTCAGCCCGATTC CGTTACCTAATGGGGGAAAGGCTGGGAGTTCACCCATTAAGCTGTCATGGGTGGGT CCTTGGGGAACATGGAGATTCCAGTGTGCCTGTATGGAGTGGAATGAATGTTGCTG GTGTCTCTCTGAAGACTCTGCACCCAGATTTAGGGACTGATAAAGATAAGGAACAG TGGAAAGAGGTTCACAAGCAGGTGGTTGAGAGTGCTTATGAGGTGATCAAACTCAA AGGCTACACATCCTGGGCTATTGGACTCTCTGTAGCAGATTTGGCAGAGAGTATAA TGAAGAATCTTAGGCGGGTGCACCCAGTTTCCACCATGATTAAGGGTCTTTACGGA ATAAAGGATGATGTCTTCCTTAGTGTTCCTTGCATTTTGGGACAGAATGGAATCTC AGACCTTGTGAAGGTGACTCTGACTTCTGAGGAAGAGGCCCGTTTGAAGAAGAGTG CAGATACACTTTGGGGGATCCAAAAGGAGCTGCAATTTTAA 1232 ATGGGTGAACCCTCAGGAGGCTATACTTACACCCAAACGTCGATATTCCTTTTCCA LDHA CGCTAAGATTCCTTTTGGTTCCAAGTCCAATATGGCAACTCTAAAGGATCAGCTGA isoform 3 TTTATAATCTTCTAAAGGAAGAACAGACCCCCCAGAATAAGATTACAGTTGTTGGG cDNA GTTGGTGCTGTTGGCATGGCCTGTGCCATCAGTATCTTAATGAAGGACTTGGCAGA TGAACTTGCTCTTGTTGATGTCATCGAAGACAAATTGAAGGGAGAGATGATGGATC TCCAACATGGCAGCCTTTTCCTTAGAACACCAAAGATTGTCTCTGGCAAAGACTAT AATGTAACTGCAAACTCCAAGCTGGTCATTATCACGGCTGGGGCACGTCAGCAAGA GGGAGAAAGCCGTCTTAATTTGGTCCAGCGTAACGTGAACATCTTTAAATTCATCA TTCCTAATGTTGTAAAATACAGCCCGAACTGCAAGTTGCTTATTGTTTCAAATCCA GTGGATATCTTGACCTACGTGGCTTGGAAGATAAGTGGTTTTCCCAAAAACCGTGT TATTGGAAGCGGTTGCAATCTGGATTCAGCCCGATTCCGTTACCTAATGGGGGAAA GGCTGGGAGTTCACCCATTAAGCTGTCATGGGTGGGTCCTTGGGGAACATGGAGAT TCCAGTGTGCCTGTATGGAGTGGAATGAATGTTGCTGGTGTCTCTCTGAAGACTCT GCACCCAGATTTAGGGACTGATAAAGATAAGGAACAGTGGAAAGAGGTTCACAAGC AGGTGGTTGAGAGTGCTTATGAGGTGATCAAACTCAAAGGCTACACATCCTGGGCT ATTGGACTCTCTGTAGCAGATTTGGCAGAGAGTATAATGAAGAATCTTAGGCGGGT GCACCCAGTTTCCACCATGATTAAGGGTCTTTACGGAATAAAGGATGATGTCTTCC TTAGTGTTCCTTGCATTTTGGGACAGAATGGAATCTCAGACCTTGTGAAGGTGACT CTGACTTCTGAGGAAGAGGCCCGTTTGAAGAAGAGTGCAGATACACTTTGGGGGAT CCAAAAGGAGCTGCAATTTTAA 1233 ATGGCAACTCTAAAGGATCAGCTGATTTATAATCTTCTAAAGGAAGAACAGACCCC LDHA CCAGAATAAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATGGCCTGTGCCATCA isoform 4 GTATCTTAATGAAGGACTTGGCAGATGAACTTGCTCTTGTTGATGTCATCGAAGAC cDNA AAATTGAAGGGAGAGATGATGGATCTCCAACATGGCAGCCTTTTCCTTAGAACACC AAAGATTGTCTCTGGCAAAGACTATAATGTAACTGCAAACTCCAAGCTGGTCATTA TCACGGCTGGGGCACGTCAGCAAGAGGGAGAAAGCCGTCTTAATTTGGTCCAGCGT AACGTGAACATCTTTAAATTCATCATTCCTAATGTTGTAAAATACAGCCCGAACTG CAAGTTGCTTATTGTTTCAAATCCAGTGGATATCTTGACCTACGTGGCTTGGAAGA TAAGTGGTTTTCCCAAAAACCGTGTTATTGGAAGCGGTTGCAATCTGGATTCAGCC CGATTCCGTTACCTAATGGGGGAAAGGCTGGGAGTTCACCCATTAAGCTGTCATGG GTGGGTCCTTGGGGAACATGGAGATTCCAGTGTGCCTGTATGGAGTGGAATGAATG TTGCTGGTGTCTCTCTGAAGACTCTGCACCCAGATTTAGGGACTGATAAAGATAAG GAACAGTGGAAAGAGTGCAGATACACTTTGGGGGATCCAAAAGGAGCTGCAATTTT AAAGTCTTCTGATGTCATATCATTTCACTGTCTAGGCTACAACAGGATTCTAGGTG GAGGTTGTGCATGTTGTCCTTTTTATCTGATCTGTGATTAA 1234 ATGGCAACTCTAAAGGATCAGCTGATTTATAATCTTCTAAAGGAAGAACAGACCCC LDHA CCAGAATAAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATGGCCTGTGCCATCA isoform 4 GTATCTTAATGAAGGACTTGGCAGATGAACTTGCTCTTGTTGATGTCATCGAAGAC cDNA AAATTGAAGGGAGAGATGATGGATCTCCAACATGGCAGCCTTTTCCTTAGAACACC AAAGATTGTCTCTGGCAAAGACTATAATGTAACTGCAAACTCCAAGCTGGTCATTA TCACGGCTGGGGCACGTCAGCAAGAGGGAGAAAGCCGTCTTAATTTGGTCCAGCGT AACGTGAACATCTTTAAATTCATCATTCCTAATGTTGTAAAATACAGCCCGAACTG CAAGTTGCTTATTGTTTCAAATCCAGTGGATATCTTGACCTACGTGGCTTGGAAGA TAAGTGGTTTTCCCAAAAACCGTGTTATTGGAAGCGGTTGCAATCTGGATTCAGCC CGATTCCGTTACCTAATGGGGGAAAGGCTGGGAGTTCACCCATTAAGCTGTCATGG GTGGGTCCTTGGGGAACATGGAGATTCCAGTGTGCCTGTATGGAGTGGAATGAATG TTGCTGGTGTCTCTCTGAAGACTCTGCACCCAGATTTAGGGACTGATAAAGATAAG GAACAGTGGAAAGAGGTTCACAAGCAGGTGGTTGAGAGGGTCTTTACGGAATAA 1254 rArGrArArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrArGrGrA 3′ end rCrUrUrGrGrCrArGrArUrGmA*mA*mC*rU modified RNA guide targeting LDHA sequence of SEQ ID NO: 1237 1255 mA*mG*mA*rArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrArGr 5′ and 3′ GrArCrUrUrGrGrCrArGrArUrGmA*mA*mC*rU end modified RNA guide targeting LDHA sequence of SEQ ID NO: 1237 1256 rArGrArArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrGrArUrGrA 3′ end rCrArUrCrArArCrArArGrAmG*mC*mA*rA modified RNA guide targeting LDHA sequence of SEQ ID NO: 1239 1257 mA*mG*mA*rArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrGrArUr 5′ and 3′ GrArCrArUrCrArArCrArArGrAmG*mC*mA*rA end modified RNA guide targeting LDHA sequence of SEQ ID NO: 1239 1258 rArGrArArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrCrArUrA 3′ end rGrUrGrGrArUrArUrCrUrUmG*mA*mC*rC modified RNA guide targeting LDHA sequence of SEQ ID NO: 1248 1259 mA*mG*mA*rArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrCrAr 5′ and 3′ UrArGrUrGrGrArUrArUrCrUrUmG*mA*mC*rC end modified RNA guide targeting LDHA sequence of SEQ ID NO: 1248 1260 rArGrArArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrUrCrArU 3′ end rArGrUrGrGrArUrArUrCrUmU*mG*mA*rC modified RNA guide targeting LDHA sequence of SEQ ID NO: 1245 1261 mA*mG*mA*rArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrUrCr 5′ and 3′ ArUrArGrUrGrGrArUrArUrCrUmU*mG*mA*rC end modified RNA guide targeting LDHA sequence of SEQ ID NO: 1245 1262 rArGrArArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrCrArUrArG 3′ end rUrGrGrArUrArUrCrUrUrGmA*mC*mC*rU modified RNA guide targeting LDHA sequence of SEQ ID NO: 1249 1263 mA*mG*mA*rArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrCrArUr 5′ and 3′ ArGrUrGrGrArUrArUrCrUrUrGmA*mC*mC*rU end modified RNA guide targeting LDHA sequence of SEQ ID NO: 1249

In some embodiments, the gene editing system disclosed herein may comprise a Cas12i polypeptide as disclosed herein. In other embodiments, the gene editing system may comprise a nucleic acid encoding the Cas12i polypeptide. For example, the gene editing system may comprise a vector (e.g., a viral vector such as an AAV vector, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11 and AAV12) encoding the Cas12i polypeptide. Alternatively, the gene editing system may comprise a mRNA molecule encoding the Cas12i polypeptide. In some instances, the mRNA molecule may be codon-optimized.

II. Preparation of Gene Editing System Components

The present disclosure provides methods for production of components of the gene editing systems disclosed herein, e.g., the RNA guide, methods for production of the Cas12i polypeptide, and methods for complexing the RNA guide and Cas12i polypeptide.

A. RNA Guide

In some embodiments, the RNA guide is made by in vitro transcription of a DNA template. Thus, for example, in some embodiments, the RNA guide is generated by in vitro transcription of a DNA template encoding the RNA guide using an upstream promoter sequence (e.g., a T7 polymerase promoter sequence).

In some embodiments, the DNA template encodes multiple RNA guides or the in vitro transcription reaction includes multiple different DNA templates, each encoding a different RNA guide. In some embodiments, the RNA guide is made using chemical synthetic methods. In some embodiments, the RNA guide is made by expressing the RNA guide sequence in cells transfected with a plasmid including sequences that encode the RNA guide. In some embodiments, the plasmid encodes multiple different RNA guides. In some embodiments, multiple different plasmids, each encoding a different RNA guide, are transfected into the cells. In some embodiments, the RNA guide is expressed from a plasmid that encodes the RNA guide and also encodes a Cas12i polypeptide. In some embodiments, the RNA guide is expressed from a plasmid that expresses the RNA guide but not a Cas12i polypeptide. In some embodiments, the RNA guide is purchased from a commercial vendor. In some embodiments, the RNA guide is synthesized using one or more modified nucleotide, e.g., as described above.

B. Cas12i Polypeptide

In some embodiments, the Cas12i polypeptide of the present disclosure can be prepared by (a) culturing bacteria which produce the Cas12i polypeptide of the present disclosure, isolating the Cas12i polypeptide, optionally, purifying the Cas12i polypeptide, and complexing the Cas12i polypeptide with an RNA guide. The Cas12i polypeptide can be also prepared by (b) a known genetic engineering technique, specifically, by isolating a gene encoding the Cas12i polypeptide of the present disclosure from bacteria, constructing a recombinant expression vector, and then transferring the vector into an appropriate host cell that expresses the RNA guide for expression of a recombinant protein that complexes with the RNA guide in the host cell. Alternatively, the Cas12i polypeptide can be prepared by (c) an in vitro coupled transcription-translation system and then complexing with an RNA guide.

In some embodiments, a host cell is used to express the Cas12i polypeptide. The host cell is not particularly limited, and various known cells can be preferably used. Specific examples of the host cell include bacteria such as E. coli, yeasts (budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (for example, CHO cells, COS cells and HEK293 cells). The method for transferring the expression vector described above into host cells, i.e., the transformation method, is not particularly limited, and known methods such as electroporation, the calcium phosphate method, the liposome method and the DEAE dextran method can be used.

After a host is transformed with the expression vector, the host cells may be cultured, cultivated or bred, for production of the Cas12i polypeptide. After expression of the Cas12i polypeptide, the host cells can be collected and Cas12i polypeptide purified from the cultures etc. according to conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.).

In some embodiments, the methods for Cas12i polypeptide expression comprises translation of at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids, or at least 1000 amino acids of the Cas12i polypeptide. In some embodiments, the methods for protein expression comprises translation of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 50 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, about 700 amino acids, about 800 amino acids, about 900 amino acids, about 1000 amino acids or more of the Cas12i polypeptide.

A variety of methods can be used to determine the level of production of a Cas12i polypeptide in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the Cas12i polypeptide or a labeling tag as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See, e.g., Maddox et al., J. Exp. Med. 158:1211 [1983]).

The present disclosure provides methods of in vivo expression of the Cas12i polypeptide in a cell, comprising providing a polyribonucleotide encoding the Cas12i polypeptide to a host cell wherein the polyribonucleotide encodes the Cas12i polypeptide, expressing the Cas12i polypeptide in the cell, and obtaining the Cas12i polypeptide from the cell.

The present disclosure further provides methods of in vivo expression of a Cas12i polypeptide in a cell, comprising providing a polyribonucleotide encoding the Cas12i polypeptide to a host cell wherein the polyribonucleotide encodes the Cas12i polypeptide and expressing the Cas12i polypeptide in the cell. In some embodiments, the polyribonucleotide encoding the Cas12i polypeptide is delivered to the cell with an RNA guide and, once expressed in the cell, the Cas12i polypeptide and the RNA guide form a complex. In some embodiments, the polyribonucleotide encoding the Cas12i polypeptide and the RNA guide are delivered to the cell within a single composition. In some embodiments, the polyribonucleotide encoding the Cas12i polypeptide and the RNA guide are comprised within separate compositions. In some embodiments, the host cell is present in a subject, e.g., a human patient.

C. Complexing

In some embodiments, an RNA guide targeting LDHA is complexed with a Cas12i polypeptide to form a ribonucleoprotein. In some embodiments, complexation of the RNA guide and Cas12i polypeptide occurs at a temperature lower than about any one of 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 50° C., or 55° C. In some embodiments, the RNA guide does not dissociate from the Cas12i polypeptide at about 37° C. over an incubation period of at least about any one of 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, 35 mins, 40 mins, 45 mins, 50 mins, 55 mins, 1 hr, 2 hr, 3 hr, 4 hr, or more hours.

In some embodiments, the RNA guide and Cas12i polypeptide are complexed in a complexation buffer. In some embodiments, the Cas12i polypeptide is stored in a buffer that is replaced with a complexation buffer to form a complex with the RNA guide. In some embodiments, the Cas12i polypeptide is stored in a complexation buffer.

In some embodiments, the complexation buffer has a pH in a range of about 7.3 to 8.6. In one embodiment, the pH of the complexation buffer is about 7.3. In one embodiment, the pH of the complexation buffer is about 7.4. In one embodiment, the pH of the complexation buffer is about 7.5. In one embodiment, the pH of the complexation buffer is about 7.6. In one embodiment, the pH of the complexation buffer is about 7.7. In one embodiment, the pH of the complexation buffer is about 7.8. In one embodiment, the pH of the complexation buffer is about 7.9. In one embodiment, the pH of the complexation buffer is about 8.0. In one embodiment, the pH of the complexation buffer is about 8.1. In one embodiment, the pH of the complexation buffer is about 8.2. In one embodiment, the pH of the complexation buffer is about 8.3. In one embodiment, the pH of the complexation buffer is about 8.4. In one embodiment, the pH of the complexation buffer is about 8.5. In one embodiment, the pH of the complexation buffer is about 8.6.

In some embodiments, the Cas12i polypeptide can be overexpressed and complexed with the RNA guide in a host cell prior to purification as described herein. In some embodiments, mRNA or DNA encoding the Cas12i polypeptide is introduced into a cell so that the Cas12i polypeptide is expressed in the cell. In some embodiments, the RNA guide is also introduced into the cell, whether simultaneously, separately, or sequentially from a single mRNA or DNA construct, such that the ribonucleoprotein complex is formed in the cell.

III. Genetic Editing Methods

The disclosure also provides methods of modifying a target site within the LDHA gene. In some embodiments, the methods comprise introducing an LDHA-targeting RNA guide and a Cas12i polypeptide into a cell. The LDHA-targeting RNA guide and Cas12i polypeptide can be introduced as a ribonucleoprotein complex into a cell. The LDHA-targeting RNA guide and Cas12i polypeptide can be introduced on a nucleic acid vector. The Cas12i polypeptide can be introduced as an mRNA. The RNA guide can be introduced directly into the cell. In some embodiments, the composition described herein is delivered to a cell/tissue/liver/person to reduce LDHA in the cell/tissue/liver/person. In some embodiments, the composition described herein is delivered to a cell/tissue/liver/person to reduce oxalate production in the cell/tissue/liver/person. In some embodiments, the composition described herein is delivered to a cell/tissue/liver/person to correct calcium oxalate crystal deposition in the cell/tissue/liver/person. In some embodiments, the composition described herein is delivered to a person with primary hyperoxaluria.

Any of the gene editing systems disclosed herein may be used to genetically engineered an LDHA gene. The gene editing system may comprise a RNA guide and a Cas12i2 polypeptide. The RNA guide comprises a spacer sequence specific to a target sequence in the LDHA gene, e.g., specific to a region in exon 3 or exon 5 of the LDHA gene.

A. Target Sequence

In some embodiments, an RNA guide as disclosed herein is designed to be complementary to a target sequence that is adjacent to a 5′-TTN-3′ PAM sequence or 5′-NTTN-3′ PAM sequence.

In some embodiments, the target sequence is within an LDHA gene or a locus of an LDHA gene (e.g., exon 3 or exon 5), to which the RNA guide can bind via base pairing. In some embodiments, a cell has only one copy of the target sequence. In some embodiments, a cell has more than one copy, such as at least about any one of 2, 3, 4, 5, 10, 100, or more copies of the target sequence.

In some embodiments, the LDHA gene is a mammalian gene. In some embodiments, the LDHA gene is a human gene. For example, in some embodiments, the target sequence is within the sequence of SEQ ID NO: 1172 (or the reverse complement thereof). In some embodiments, the target sequence is within an exon of the LDHA gene set forth in SEQ ID NO: 1172, e.g., within a sequence of SEQ ID NO: 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, or 1181 (or a reverse complement thereof). Target sequences within an exon region of the LDHA gene of SEQ ID NO: 1172 are set forth in Table 5. In some embodiments, the target sequence is within an intron of the LDHA gene set forth in SEQ ID NO: 1172 (or the reverse complement thereof). In some embodiments, the target sequence is within a variant (e.g., a polymorphic variant) of the LDHA gene sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof). In some embodiments, the LDHA gene sequence is a homolog of the sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof). For examples, in some embodiments, the LDHA gene sequence is a non-human LDHA sequence. In some embodiments, the LDHA gene sequence is a coding sequence set forth in any one of SEQ ID NOs: 1230-1234 (or the reverse complement thereof). In some embodiments, the LDHA gene sequence is a homolog of a coding sequence set forth in any one of SEQ ID NOs: 1230-1234 (or the reverse complement thereof).

In some embodiments, the target sequence is adjacent to a 5′-NTTN-3′ PAM sequence or 5′-TTN-3′ PAM sequence, wherein N is any nucleotide. The 5′-NTTN-3′ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence. In some embodiments the 5′-NTTN-3′ sequence is 5′-NTTY-3′, 5′-NTTC-3′, 5′-NTTT-3′, 5′-NTTA-3′, 5′-NTTB-3′, 5′-NTTG-3′, 5′-CTTY-3′, 5′-DTTR-3′, 5′-CTTR-3′, 5′-DTTT-3′, 5′-ATTN-3′, or 5′-GTTN-3′, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G. In some embodiments, the 5′-NTTN-3′ sequence is 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′. The PAM sequence may be 5′ to the target sequence.

The 5′-NTTN-3′ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence. In some embodiments the 5′-NTTN-3′ sequence is 5′-NTTY-3′, 5′-NTTC-3′, 5′-NTTT-3′, 5′-NTTA-3′, 5′-NTTB-3′, 5′-NTTG-3′, 5′-CTTY-3′, 5′-DTTR-3′, 5′-CTTR-3′, 5′-DTTT-3′, 5′-ATTN-3′, or 5′-GTTN-3′, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G. In some embodiments, the 5′-NTTN-3′ sequence is 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′. In some embodiments, the RNA guide is designed to bind to a first strand of a double-stranded target nucleic acid (i.e., the non-PAM strand), and the 5′-NTTN-3′ PAM sequence is present in the second, complementary strand (i.e., the PAM strand). In some embodiments, the RNA guide binds to a region on the non-PAM strand that is complementary to a target sequence on the PAM strand, which is adjacent to a 5′-NAAN-3′ sequence.

In some embodiments, the target sequence is present in a cell. In some embodiments, the target sequence is present in the nucleus of the cell. In some embodiments, the target sequence is endogenous to the cell. In some embodiments, the target sequence is a genomic DNA. In some embodiments, the target sequence is a chromosomal DNA. In some embodiments, the target sequence is a protein-coding gene or a functional region thereof, such as a coding region, or a regulatory element, such as a promoter, enhancer, a 5′ or 3′ untranslated region, etc.

In some embodiments, the target sequence is present in a readily accessible region of the target sequence. In some embodiments, the target sequence is in an exon of a target gene. In some embodiments, the target sequence is across an exon-intron junction of a target gene. In some embodiments, the target sequence is present in a non-coding region, such as a regulatory region of a gene.

B. Gene Editing

In some embodiments, the Cas12i polypeptide has enzymatic activity (e.g., nuclease activity). In some embodiments, the Cas12i polypeptide induces one or more DNA double-stranded breaks in the cell. In some embodiments, the Cas12i polypeptide induces one or more DNA single-stranded breaks in the cell. In some embodiments, the Cas12i polypeptide induces one or more DNA nicks in the cell. In some embodiments, DNA breaks and/or nicks result in formation of one or more indels (e.g., one or more deletions).

In some embodiments, an RNA guide disclosed herein forms a complex with the Cas12i polypeptide and directs the Cas12i polypeptide to a target sequence adjacent to a 5′-NTTN-3′ sequence. In some embodiments, the complex induces a deletion (e.g., a nucleotide deletion or DNA deletion) adjacent to the 5′-NTTN-3′ sequence. In some embodiments, the complex induces a deletion adjacent to a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the complex induces a deletion adjacent to a T/C-rich sequence.

In some embodiments, the deletion is downstream of a 5′-NTTN-3′ sequence. In some embodiments, the deletion is downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion is downstream of a T/C-rich sequence.

In some embodiments, the deletion alters expression of the LDHA gene. In some embodiments, the deletion alters function of the LDHA gene. In some embodiments, the deletion inactivates the LDHA gene. In some embodiments, the deletion is a frameshifting deletion. In some embodiments, the deletion is a non-frameshifting deletion. In some embodiments, the deletion leads to cell toxicity or cell death (e.g., apoptosis).

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion is up to about 40 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides). In some embodiments, the deletion is between about 4 nucleotides and about 40 nucleotides in length (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides). In some embodiments, the deletion is between about 4 nucleotides and about 25 nucleotides in length (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 25 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 15 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides).

In some embodiments, the methods described herein are used to engineer a cell comprising a deletion as described herein in an LDHA gene. In some embodiments, the methods are carried out using a complex comprising a Cas12i enzyme as described herein and an RNA guide comprising a direct repeat and a spacer as described herein. In some embodiments, the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229. In some embodiments, an RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229. In some embodiments, the RNA guide targeting LDHA is encoded in a plasmid. In some embodiments, the RNA guide targeting LDHA is synthetic or purified RNA. In some embodiments, the Cas12i polypeptide is encoded in a plasmid. In some embodiments, the Cas12i polypeptide is encoded by an RNA that is synthetic or purified.

C. Delivery

Components of any of the gene editing systems disclosed herein may be formulated, for example, including a carrier, such as a carrier and/or a polymeric carrier, e.g., a liposome, and delivered by known methods to a cell (e.g., a prokaryotic, eukaryotic, plant, mammalian, etc.). Such methods include, but not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)), microinjection, microprojectile bombardment (“gene gun”), fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof.

In some embodiments, the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding the Cas12i polypeptide, RNA guide, donor DNA, etc.), one or more transcripts thereof, and/or a pre-formed RNA guide/Cas12i polypeptide complex to a cell, where a ternary complex is formed. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide are delivered together in a single composition. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide are delivered in separate compositions. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide delivered in separate compositions are delivered using the same delivery technology. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide delivered in separate compositions are delivered using different delivery technologies. Exemplary intracellular delivery methods, include, but are not limited to: viruses, such as AAV, or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, lipid nanoparticles, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection. In some embodiments, a lipid nanoparticle comprises an mRNA encoding a Cas12i polypeptide, an RNA guide, or an mRNA encoding a Cas12i polypeptide and an RNA guide. In some embodiments, the mRNA encoding the Cas12i polypeptide is a transcript of the nucleotide sequence set forth in SEQ ID NO: 1165 or SEQ ID NO: 1201 or a variant thereof. In some embodiments, the present application further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.

D. Genetically Modified Cells

Any of the gene editing systems disclosed herein can be delivered to a variety of cells. In some embodiments, the cell is an isolated cell. In some embodiments, the cell is in cell culture or a co-culture of two or more cell types. In some embodiments, the cell is ex vivo. In some embodiments, the cell is obtained from a living organism and maintained in a cell culture. In some embodiments, the cell is a single-cellular organism.

In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell or derived from a bacterial cell. In some embodiments, the cell is an archaeal cell or derived from an archaeal cell.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a plant cell or derived from a plant cell. In some embodiments, the cell is a fungal cell or derived from a fungal cell. In some embodiments, the cell is an animal cell or derived from an animal cell. In some embodiments, the cell is an invertebrate cell or derived from an invertebrate cell. In some embodiments, the cell is a vertebrate cell or derived from a vertebrate cell. In some embodiments, the cell is a mammalian cell or derived from a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebra fish cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is synthetically made, sometimes termed an artificial cell.

In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cell is an immortal or immortalized cell.

In some embodiments, the cell is a primary cell. In some embodiments, the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC. In some embodiments, the cell is a differentiated cell. For example, in some embodiments, the differentiated cell is a liver cell (e.g., a hepatocyte), a biliary cell (e.g., a cholangiocyte), a stellate cell, a Kupffer cell, a liver sinusoidal endothelial cell, a muscle cell (e.g., a myocyte), a fat cell (e.g., an adipocyte), a bone cell (e.g., an osteoblast, osteocyte, osteoclast), a blood cell (e.g., a monocyte, a lymphocyte, a neutrophil, an eosinophil, a basophil, a macrophage, a erythrocyte, or a platelet), a nerve cell (e.g., a neuron), an epithelial cell, an immune cell (e.g., a lymphocyte, a neutrophil, a monocyte, or a macrophage), a fibroblast, or a sex cell. In some embodiments, the cell is a terminally differentiated cell. For example, in some embodiments, the terminally differentiated cell is a neuronal cell, an adipocyte, a cardiomyocyte, a skeletal muscle cell, an epidermal cell, or a gut cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a Natural Killer (NK) cell. In some embodiments, the immune cell is a Tumor Infiltrating Lymphocyte (TIL). In some embodiments, the cell is a mammalian cell, e.g., a human cell or a murine cell. In some embodiments, the murine cell is derived from a wild-type mouse, an immunosuppressed mouse, or a disease-specific mouse model. In some embodiments, the cell is a cell within a living tissue, organ, or organism.

Any of the genetically modified cells produced using any of the gene editing system disclosed herein is also within the scope of the present disclosure. Such modified cells may comprise a disrupted LDHA gene.

Compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in therapy. Compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in methods of treating a disease or condition in a subject. In some embodiments, the disease or condition is primary hyperoxaluria (PH). In some embodiments, the PH is PH1, PH2, or PH3. Any suitable delivery or administration method known in the art may be used to deliver compositions, vectors, nucleic acids, RNA guides and cells disclosed herein. Such methods may involve contacting a target sequence with a composition, vector, nucleic acid, or RNA guide disclosed herein. Such methods may involve a method of editing an LDHA sequence as disclosed herein. In some embodiments, a cell engineered using an RNA guide disclosed herein is used for ex vivo gene therapy.

IV. Therapeutic Applications

Any of the gene editing systems or modified cells generated using such a gene editing system as disclosed herein may be used for treating a disease that is associated with the LDHA gene, for example, primary hyperoxaluria (PH). In some embodiments, the PH is PH1, PH2, or PH3. In specific examples, the target disease is PH1.

PH is a rare genetic disorder effecting subjects of all ages from infants to elderly. PH includes three subtypes involving genetic defects that alter the expression of three distinct proteins. PH1 involves alanine-glyoxylate aminotransferase, or AGT/AGT1. PH2 involves glyoxylate/hydroxypyruvate reductase, or GR/HPR, and PH3 involves 4-hydroxy-2-oxoglutarate aldolase, or HOGA.

In PH1, excess oxalate can also combine with calcium to form calcium oxalate in the kidney and other organs. Deposits of calcium oxalate can produce widespread deposition of calcium oxalate (nephrocalcinosis) or formation of kidney and bladder stones (urolithiasis) and lead to kidney damage. Common kidney complications in PH1 include blood in the urine (hematuria), urinary tract infections, kidney damage, and end-stage renal disease (ESRD). Over time, kidneys in patients with PH1 may begin to fail, and levels of oxalate may rise in the blood. Deposition of oxalate in tissues throughout the body, e.g., systemic oxalosis, may occur due to high blood levels of oxalate and can lead to complications in bone, skin, and eye. Patients with PH1 normally have kidney failure at an early age, with renal dialysis or dual kidney/liver organ transplant as the only treatment options.

In some embodiments, provided herein is a method for treating a target disease as disclosed herein (e.g., PH such as PH1) comprising administering to a subject (e.g., a human patient) in need of the treatment any of the gene editing systems disclosed herein. The gene editing system may be delivered to a specific tissue or specific type of cells where the gene edit is needed. The gene editing system may comprise LNPs encompassing one or more of the components, one or more vectors (e.g., viral vectors) encoding one or more of the components, or a combination thereof. Components of the gene editing system may be formulated to form a pharmaceutical composition, which may further comprise one or more pharmaceutically acceptable carriers.

In some embodiments, modified cells produced using any of the gene editing systems disclosed herein may be administered to a subject (e.g., a human patient) in need of the treatment. The modified cells may comprise a substitution, insertion, and/or deletion described herein. In some examples, the modified cells may include a cell line modified by a CRISPR nuclease, reverse transcriptase polypeptide, and editing template RNA (e.g., RNA guide and RT donor RNA). In some instances, the modified cells may be a heterogenous population comprising cells with different types of gene edits. Alternatively, the modified cells may comprise a substantially homogenous cell population (e.g., at least 80% of the cells in the whole population) comprising one particular gene edit in the LDHA gene. In some examples, the cells can be suspended in a suitable media.

In some embodiments, provided herein is a composition comprising the gene editing system or components thereof. Such a composition can be a pharmaceutical composition. A pharmaceutical composition that is useful may be prepared, packaged, or sold in a formulation suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, intra-lesional, buccal, ophthalmic, intravenous, intra-organ or another route of administration. A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition (e.g., the gene editing system or components thereof), which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

In some embodiments, a pharmaceutical composition comprising the gene editing system or components thereof as described herein may be administered to a subject in need thereof, e.g., one who suffers from a liver disease associated with the LDHA gene. In some instances, the gene editing system or components thereof may be delivered to specific cells or tissue (e.g., to liver cells), where the gene editing system could function to genetically modify the LDHA gene in such cells.

A formulation of a pharmaceutical composition suitable for parenteral administration may comprise the active agent (e.g., the gene editing system or components thereof or the modified cells) combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Some injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Some formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the cells, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulation may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or saline. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which that are useful include those which may comprise the cells in a packaged form, in a liposomal preparation, or as a component of a biodegradable polymer system. Some compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

V. Kits and Uses Thereof

The present disclosure also provides kits that can be used, for example, to carry out a method described herein for genetical modification of the LDHA gene. In some embodiments, the kits include an RNA guide and a Cas12i polypeptide. In some embodiments, the kits include a polynucleotide that encodes such a Cas12i polypeptide, and optionally the polynucleotide is comprised within a vector, e.g., as described herein. The Cas12i polypeptide and the RNA guide (e.g., as a ribonucleoprotein) can be packaged within the same or other vessel within a kit or system or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The kits can additionally include, optionally, a buffer and/or instructions for use of the RNA guide and Cas12i polypeptide.

In some embodiments, the kit may be useful for research purposes. For example, in some embodiments, the kit may be useful to study gene function.

All references and publications cited herein are hereby incorporated by reference.

Additional Embodiments

Provided below are additional embodiments, which are also within the scope of the present disclosure.

Embodiment 1: A composition comprising an RNA guide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary or complete complementary to a region on a non-PAM strand (the complementary sequence of a target sequence) within an LDHA gene and (ii) a direct repeat sequence; wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′.

In Embodiment 1, the target sequence may be within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or exon 9 of the LDHA gene. In some examples, the LDHA gene comprises the sequence of SEQ ID NO: 1172, the reverse complement of SEQ ID NO: 1172, a variant of SEQ ID NO: 1172, or the reverse complement of a variant of SEQ ID NO: 1172.

In Embodiment 1, the spacer sequence may comprise: (a) nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164.

In any of the compositions of Embodiment 1, the spacer sequence may comprise: (a) nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 588-1164.

In any of the compositions of Embodiment 1, the direct repeat sequence may comprise: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; or (aa) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (1) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; or (aa) SEQ ID NO: 10 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1200 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; or (o) SEQ ID NO: 1200 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1205; or (o) SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1210 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) SEQ ID NO: 1210 or a portion thereof.

In some examples, the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-587.

In any of the composition of Embodiment 1, the PAM may comprise the sequence 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.

In some examples, the target sequence is immediately adjacent to the PAM sequence.

In some examples, the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1213-1229.

In some examples, the RNA guide has the sequence of any one of SEQ ID NOs: 1213-1229.

Embodiment 2: The composition of Embodiment 1 may further comprise a Cas12i polypeptide or a polyribonucleotide encoding a Cas12i polypeptide, which can be one of the following: (a) a Cas12i2 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1212.

In specific examples, the Cas12i polypeptide is: (a) a Cas12i2 polypeptide comprising a sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence of SEQ ID NO: 1212.

In any of the compositions of Embodiment 2, the RNA guide and the Cas12i polypeptide may form a ribonucleoprotein complex. In some examples, the ribonucleoprotein complex binds a target nucleic acid. In some examples, the composition is present within a cell.

In any of the compositions of Embodiment 2, the RNA guide and the Cas12i polypeptide may be encoded in a vector, e.g., expression vector. In some examples, the RNA guide and the Cas12i polypeptide are encoded in a single vector. In other examples, the RNA guide is encoded in a first vector and the Cas12i polypeptide is encoded in a second vector.

Embodiment 3: A vector system comprising one or more vectors encoding an RNA guide disclosed herein and a Cas12i polypeptide. In some examples, the vector system comprises a first vector encoding an RNA guide disclosed herein and a second vector encoding a Cas12i polypeptide. The vectors may be expression vectors.

Embodiment 4: A composition comprising an RNA guide and a Cas12i polypeptide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary or completely complementary to a region on a non-PAM strand (the complementary sequence of a target sequence) within an LDHA gene, and (ii) a direct repeat sequence.

In some examples, the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or exon 9 of the LDHA gene, which may comprise the sequence of SEQ ID NO: 1172, the reverse complement of SEQ ID NO: 1172, a variant of the sequence of SEQ ID NO: 1172, or the reverse complement of a variant of SEQ ID NO: 1172.

In some examples, the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164.

In some examples, the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 588-1164.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; or (aa) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (1) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; or (aa) SEQ ID NO: 10 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1200 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; or (o) SEQ ID NO: 1200 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1205; or (o) SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1210 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) SEQ ID NO: 1210 or a portion thereof.

In any of the compositions of Embodiment 4, the spacer sequence may be substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-587.

In some examples, the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′. In some examples, the PAM comprises the sequence 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.

In some examples, the target sequence is immediately adjacent to the PAM sequence. In some examples, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.

In any of the compositions of Embodiment 4, the Cas12i polypeptide is: (a) a Cas12i2 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1212.

In some examples, the Cas12i polypeptide is: (a) a Cas12i2 polypeptide comprising a sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence of SEQ ID NO: 1212.

In any of the composition of Embodiment 4, the RNA guide and the Cas12i polypeptide may form a ribonucleoprotein complex. In some examples, the ribonucleoprotein complex binds a target nucleic acid.

In any of the composition of Embodiment 4, the composition may be present within a cell.

In any of the composition of Embodiment 4, the RNA guide and the Cas12i polypeptide may be encoded in a vector, e.g., expression vector. In some examples, the RNA guide and the Cas12i polypeptide are encoded in a single vector. In other examples, the RNA guide is encoded in a first vector and the Cas12i polypeptide is encoded in a second vector.

Embodiment 5: A vector system comprising one or more vectors encoding an RNA guide disclosed herein and a Cas12i polypeptide. In some examples, the vector system comprises a first vector encoding an RNA guide disclosed herein and a second vector encoding a Cas12i polypeptide. In some examples, the vectors are expression vectors.

Embodiment 6: An RNA guide comprising (i) a spacer sequence that is substantially complementary or completely complementary to a region on a non-PAM strand (the complementary sequence of a target sequence) within an LDHA gene, and (ii) a direct repeat sequence.

In some examples, the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or exon 9 of the LDHA gene, which may comprise the sequence of SEQ ID NO: 1172, the reverse complement of SEQ ID NO: 1172, a variant of the sequence of SEQ ID NO: 1172, or the reverse complement of a variant of SEQ ID NO: 1172.

In some examples, the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164.

In some examples, the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 588-1164.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; or (aa) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (1) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; or (aa) SEQ ID NO: 10 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1200 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; or (o) SEQ ID NO: 1200 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1205; (or o) SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1210 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) SEQ ID NO: 1210 or a portion thereof.

In any of the RNA guide of Embodiment 6, the spacer sequence may be substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-587.

In any of the RNA guide of Embodiment 6, the target sequence may be adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′, wherein N is any nucleotide. In some examples, the PAM comprises the sequence 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.

In some examples, the target sequence is immediately adjacent to the PAM sequence. In other examples, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.

In some examples, the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1213-1229. In specific examples, the RNA guide has the sequence of any one of SEQ ID NOs: 1213-1229.

Embodiment 7: A nucleic acid encoding an RNA guide as described herein.

Embodiment 8: A vector comprising such an RNA guide as described herein.

Embodiment 9: A cell comprising a composition, an RNA guide, a nucleic acid, or a vector as described herein. In some examples, the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, a primary cell, a cell line, a stem cell, or a T cell.

Embodiment 10: A kit comprising a composition, an RNA guide, a nucleic acid, or a vector as described herein.

Embodiment 11: A method of editing an LDHA sequence, the method comprising contacting an LDHA sequence with a composition or an RNA guide as described herein. In some examples, the method is carried out in vitro. In other examples, the method is carried out ex vivo.

In some examples, the LDHA sequence is in a cell.

In some examples, the composition or the RNA guide induces a deletion in the LDHA sequence. In some examples, the deletion is adjacent to a 5′-NTTN-3′ sequence, wherein N is any nucleotide. In some specific examples, the deletion is downstream of the 5′-NTTN-3′ sequence. In some specific examples, the deletion is up to about 40 nucleotides in length. In some instances, the deletion is from about 4 nucleotides to 40 nucleotides, about 4 nucleotides to 25 nucleotides, about 10 nucleotides to 25 nucleotides, or about 10 nucleotides to 15 nucleotides in length.

In some examples, the deletion starts within about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 10 nucleotides, or about 10 nucleotides to about 15 nucleotides of the 5′-NTTN-3′ sequence.

In some examples, the deletion starts within about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 10 nucleotides, or about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence.

In some examples, the deletion ends within about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 25 nucleotides, or about 25 nucleotides to about 30 nucleotides of the 5′-NTTN-3′ sequence.

In some examples, the deletion ends within about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 25 nucleotides, about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In some examples, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In some examples, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.

In some examples, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In some examples, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In some examples, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.

In some examples, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In some examples, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In some examples, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.

In some examples, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In some examples, the 5′-NTTN-3′ sequence is 5′-CTTT-3′, 5′-CTTC-3′, 5′-GTTT-3′, 5′-GTTC-3′, 5′-TTTC-3′, 5′-GTTA-3′, or 5′-GTTG-3′.

In some examples, the deletion overlaps with a mutation in the LDHA sequence. In some instances, the deletion overlaps with an insertion in the LDHA sequence. In some instances, the deletion removes a repeat expansion of the LDHA sequence or a portion thereof. In some instances, the deletion disrupts one or both alleles of the LDHA sequence.

In any of the composition, RNA guide, nucleic acid, vector, cell, kit, or method of Embodiments 1-11 described herein, the RNA guide may comprise the sequence of any one of SEQ ID NOs: 1213-1229.

Embodiment 12: A method of treating primary hyperoxaluria (PH), which optionally is PH1, PH2, or PH3, in a subject, the method comprising administering a composition, an RNA guide, or a cell described herein to the subject.

In any of the compositions, RNA guides, cells, kits, or methods described herein, the RNA guide and/or the polyribonucleotide encoding the Cas12i polypeptide are comprised within a lipid nanoparticle. In some examples, the RNA guide and the polyribonucleotide encoding the Cas12i polypeptide are comprised within the same lipid nanoparticle. In other examples, the RNA guide and the polyribonucleotide encoding the Cas12i polypeptide are comprised within separate lipid nanoparticles.

Embodiment 13: An RNA guide comprising (i) a spacer sequence that is complementary to a target site within an LDHA gene (the target site being on the non-PAM strand and complementary to a target sequence), and (ii) a direct repeat sequence, wherein the target sequence is any one of SEQ ID NOs: 1237, 1239, 1248, 1245, or 1249, or the reverse complement thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; or (aa) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (1) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; or (aa) SEQ ID NO: 10 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1200 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; or (o) SEQ ID NO: 1200 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1205; or (o) SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1210 or a portion thereof.

In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) SEQ ID NO: 1210 or a portion thereof.

In some examples, the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1214, 1235, 1224, 1221, or 1225. In specific examples, the RNA guide has the sequence of any one of SEQ ID NOs: 1214, 1235, 1224, 1221, or 1225.

In some examples, each of the first three nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification.

In some examples, each of the last four nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification.

In some examples, each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification, and wherein the last nucleotide of the RNA guide is unmodified.

In some examples, the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1254-1263. In specific examples, the RNA guide has the sequence of any one of SEQ ID NOs: 1254-1263.

In some embodiments, an LDHA-targeting RNA guide comprises at least 90% identity to any one of SEQ ID NOs: 1254-1263. In some embodiments, an LDHA-targeting RNA guide comprises any one of SEQ ID NOs: 1254-1263. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1254 or SEQ ID NO: 1255 binds the complementary region of LDHA target sequence of SEQ ID NO: 1237. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1254 or SEQ ID NO: 1255 binds the complementary region of LDHA target sequence of SEQ ID NO: 1237. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1256 or SEQ ID NO: 1257 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1239. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1256 or SEQ ID NO: 1257 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1239. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1258 or SEQ ID NO: 1259 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1248. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1258 or SEQ ID NO: 1259 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1248. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1260 or SEQ ID NO: 1261 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1245. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1260 or SEQ ID NO: 1261 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1245. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1262 or SEQ ID NO: 1263 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1249. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1262 or SEQ ID NO: 1263 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1249.

Embodiment 14: A nucleic acid encoding an RNA guide as described herein.

Embodiment 15: A vector comprising the nucleic acid as described herein.

Embodiment 16: A vector system comprising one or more vectors encoding (i) the RNA guide of Embodiment 13 as described herein and (ii) a Cas12i polypeptide. In some examples, the vector system comprises a first vector encoding the RNA guide and a second vector encoding the Cas12i polypeptide.

Embodiment 17: A cell comprising the RNA guide, the nucleic acid, the vector, or the vector system of Embodiments 13-16 as described herein. In some examples, the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, a primary cell, a cell line, a stem cell, or a T cell.

Embodiment 18: A kit comprising the RNA guide, the nucleic acid, the vector, or the vector system of Embodiments 13-16 as described herein.

Embodiment 19: A method of editing an LDHA sequence, the method comprising contacting an LDHA sequence with an RNA guide of Embodiment 13 as described herein. In some examples, the LDHA sequence is in a cell.

In some examples, the RNA guide induces an indel (e.g., an insertion or deletion) in the LDHA sequence.

Embodiment 20: A method of treating primary hyperoxaluria (PH), which optionally is PH1, PH2, or PH3, in a subject, the method comprising administering the RNA guide of Embodiment 13 as described herein to the subject.

General Techniques

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D. N. Glover ed. 1985); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985»; Transcription and Translation (B. D. Hames & S. J. Higgins, eds. (1984»; Animal Cell Culture (R. I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (1RL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.).

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES

The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of the present disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

Example 1—Cas12i2-Mediated Editing of LDHA Target Sites in HEK293T Cells

This Example describes the genomic editing of the LDHA gene using Cas12i2 introduced into HEK293T cells.

Cas12i2 RNA guides (crRNAs) were designed and ordered from Integrated DNA Technologies (IDT). For initial guide screening in HEK293T cells, target sequences were designed by tiling the coding exons of LDHA for 5′-NTTN-3′ PAM sequences, and then spacer sequences were designed for the 20-bp target sequences downstream of the PAM sequence. The LDHA-targeting RNA guide sequences are shown in Table 7. TS stands for “top strand” of the LDHA gene, and BS stands for “bottom strand” of the LDHA gene. In the figures, “E #T #” can also be represented as “exon #target #.”

TABLE 7 crRNA sequences for LDHA Target strand (non- PAM guide name PAM* strand) crRNA sequence target sequence LDHA_E2T23 CTTA TS AGAAAUCCGUCUUUCAUUG CCTTCATTAAGATA ACGGCCUUCAUUAAGAUAC CTGATG (SEQ ID UGAUG (SEQ ID NO: 1213) NO: 1236) LDHA_E3T1 CTTT BS AGAAAUCCGUCUUUCAUUG TAGGACTTGGCAG ACGGUAGGACUUGGCAGAU ATGAACT (SEQ ID GAACU (SEQ ID NO: 1214) NO: 1237) LDHA_E3T2 GTTC TS AGAAAUCCGUCUUUCAUUG ATCTGCCAAGTCCT ACGGAUCUGCCAAGUCCUA AAAAGA (SEQ ID AAAGA (SEQ ID NO: 1215) NO: 1238) LDHA_E3T3 CTTC TS AGAAAUCCGUCUUUCAUUG GATGACATCAACA ACGGGAUGACAUCAACAAG AGAGCAA (SEQ ID AGCAA (SEQ ID NO: 1235) NO: 1239) LDHA_E3T9 ATTT BS AGAAAUCCGUCUUUCAUUG GATGTCTTTTAGGA ACGGGAUGUCUUUUAGGAC CTTGGC (SEQ ID UUGGC (SEQ ID NO: 1216) NO: 1240) LDHA_E3T10 TTTG BS AGAAAUCCGUCUUUCAUUG ATGTCTTTTAGGAC ACGGAUGUCUUUUAGGACU TTGGCA (SEQ ID UGGCA (SEQ ID NO: 1217) NO: 1241) LDHA_E3T12 TTTA BS AGAAAUCCGUCUUUCAUUG GGACTTGGCAGAT ACGGGGACUUGGCAGAUGA GAACTTG (SEQ ID ACUUG (SEQ ID NO: 1218) NO: 1242) LDHA_E3T26 GTTG TS AGAAAUCCGUCUUUCAUUG AAATCAACCTTTGC ACGGAAAUCAACCUUUGCC CAGAGA (SEQ ID AGAGA (SEQ ID NO: 1219) NO: 1243) LDHA_E3T27 CTTG TS AGAAAUCCGUCUUUCAUUG TTGAAATCAACCTT ACGGUUGAAAUCAACCUUU TGCCAG (SEQ ID GCCAG (SEQ ID NO: 1220) NO: 1244) LDHA_E5T1 CTTT BS AGAAAUCCGUCUUUCAUUG TTCATAGTGGATAT ACGGUUCAUAGUGGAUAUC CTTGAC (SEQ ID UUGAC (SEQ ID NO: 1221) NO: 1245) LDHA_E5T7 TTTT BS AGAAAUCCGUCUUUCAUUG CTCCTTTTTCATAG ACGGCUCCUUUUUCAUAGU TGGATA (SEQ ID GGAUA (SEQ ID NO: 1222) NO: 1246) LDHA_E5T8 TTTC BS AGAAAUCCGUCUUUCAUUG TCCTTTTTCATAGT ACGGUCCUUUUUCAUAGUG GGATAT (SEQ ID GAU AU (SEQ ID NO: 1223) NO: 1247) LDHA_E5T9 TTTT BS AGAAAUCCGUCUUUCAUUG TCATAGTGGATATC ACGGUCAUAGUGGAUAUCU TTGACC (SEQ ID UGACC (SEQ ID NO: 1224) NO: 1248) LDHA_E5T10 TTTT BS AGAAAUCCGUCUUUCAUUG CATAGTGGATATCT ACGGCAUAGUGGAUAUCUU TGACCT (SEQ ID GACCU (SEQ ID NO: 1225) NO: 1249) LDHA_E5T11 TTTC BS AGAAAUCCGUCUUUCAUUG ATAGTGGATATCTT ACGGAUAGUGGAUAUCUUG GACCTA (SEQ ID ACCUA (SEQ ID NO: 1226) NO: 1250) LDHA_E5T28 ATTA TS AGAAAUCCGUCUUUCAUUG GGTAACGGAATCG ACGGGGUAACGGAAUCGGG GGCTGAA (SEQ ID CUGAA (SEQ ID NO: 1227) NO: 1251) LDHA_E5T32 CTTA TS AGAAAUCCGUCUUUCAUUG CCACTGGAATCTCC ACGGCCACUGGAAUCUCCA ATGTTC (SEQ ID UGUUC (SEQ ID NO: 1228) NO: 1252) LDHA_E5T33 CTTA TS AGAAAUCCGUCUUUCAUUG TGCTTACCACTGGA ACGGUGCUUACCACUGGAA ATCTCC (SEQ ID UCUCC (SEQ ID NO: 1229) NO: 1253) *The 3′ three nucleotides represent the 5′-TTN-3′ motif.

Cas12i2 RNP complexation reactions were made by mixing purified Cas12i2 polypeptide (400 μM) with crRNA (1 mM in 250 mM NaCl) at a 1:1 (Cas12i2:crRNA) volume ratio (2.5:1 crRNA:Cas12i2 molar ratio). Complexations were incubated on ice for 30-60 min.

HEK293T cells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; Thermo Fisher) and counted. Cells were washed once with PBS and resuspended in SF buffer+supplement (SF CELL LINE 4D-NUCLEOFECTOR™ X KIT S; Lonza #V4XC-2032) at a concentration of 16,480 cells/μL. Resuspended cells were dispensed at 3e5 cells/reaction into Lonza 16-well NUCLEOCUVETTE® strips. Complexed Cas12i2 RNP was added to each reaction at a final concentration of 10 μM (Cas12i2), and transfection enhancer oligos were then added at a final concentration of 4 μM. The final volume of each electroporated reaction was 20 μL. Non-targeting guides were used as negative controls.

The strips were electroporated using an electroporation device (program CM-130, Lonza 4D-NUCLEOFECTOR™). Immediately following electroporation, 80 μL of pre-warmed DMEM+10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 10 μL (30,000 cells) of diluted nucleofected cells into pre-warmed 96-well plate with wells containing 100 μL DMEM+10% FBS. Editing plates were incubated for 3 days at 37° C. with 5% CO₂.

After 3 days, wells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; Thermo Fisher) and transferred to 96-well TWIN.TEC® PCR plates (Eppendorf). Media was flicked off and cells were resuspended in 20 μL QUICKEXTRACT™ (DNA extraction buffer; Lucigen). Samples were then cycled in a PCR machine at 65° C. for 15 min, 68° C. for 15 min, 98° C. for 10 min. Samples were then frozen at −20° C.

Samples for Next Generation Sequencing (NGS) were prepared by rounds of PCR. The first round (PCR I) was used to amplify the genomic regions flanking the target site and add NGS adapters. The second round (PCR II) was used to add NGS indexes. Reactions were then pooled, purified by column purification, and quantified on a fluorometer (Qubit). Sequencing runs were done using a 150 cycle NGS instrument (NEXTSEQ™ v2.5) mid or high output kit (Illumina) and run on an NGS instrument (NEXTSEQ™ 550; Illumina).

For NGS analysis, the indel mapping function used a sample's fastq file, the amplicon reference sequence, and the forward primer sequence. For each read, a kmer-scanning algorithm was used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence. In order to remove small amounts of primer dimer present in some samples, the first 30 nt of each read was required to match the reference and reads where over half of the mapping nucleotides are mismatches were filtered out as well. Up to 50,000 reads passing those filters were used for analysis, and reads were counted as an indel read if they contained an insertion or deletion. The % indels was calculated as the number of indel-containing reads divided by the number of reads analyzed (reads passing filters up to 50,000). The QC standard for the minimum number of reads passing filters was 10,000.

FIG. 1 shows LDHA indels in HEK293T cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected within and/or adjacent to each of the LDHA target sites with each of the RNA guides. Delivery of E3T1 (SEQ ID NO: 1214), E3T9 (SEQ ID NO: 1216), EST1 (SEQ ID NO: 1221), E5T9 (SEQ ID NO: 1224), and E5T10 (SEQ ID NO: 1225) resulted in indels in over 70% of the NGS reads. Therefore, LDHA-targeting RNA guides induced indels in exon 2, exon 3, and exon 5 in HEK293T cells.

This Example thus shows that LDHA can be individually targeted by Cas12i2 RNPs in mammalian cells such as HEK293T cells.

Example 2—Cas12i2-Mediated Editing of LDHA Target Sites in Hepg2 Cells

This Example describes the genomic editing of the LDHA gene using Cas12i2 introduced into HepG2 cells by RNP.

RNP complexation reactions were performed as described in Example 1 with various RNA guides of Table 7. HepG2 cells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; ThermoFisher) and counted. Cells were washed once with PBS and resuspended in SF buffer+supplement (SF CELL LINE 4D-NUCLEOFECTOR™ X KIT S; Lonza #V4XC-2032) at a concentration of 13,889 cells/μL. Resuspended cells were dispensed at 2.5e5 cells/reaction into Lonza 16-well NUCLEOCUVETTE® strips. Complexed Cas12i2 RNP was added to each reaction at a final concentration of 20 μM (Cas12i2), with no transfection enhancer oligo. The final volume of each electroporated reaction was 20 Non-targeting guides were used as negative controls.

The strips were electroporated using an electroporation device (program DJ-100, Lonza 4D-NUCLEOFECTOR™). Immediately following electroporation, 80 μL of pre-warmed EMEM+10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 10 μL (25,000 cells) of diluted nucleofected cells into pre-warmed 96-well plate with wells containing 100 μL EMEM+10% FBS. Editing plates were incubated for 3 days at 37° C. with 5% CO₂.

After 3 days, wells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; ThermoFisher) and transferred to 96-well TWIN.TEC® PCR plates (Eppendorf). Media was flicked off and cells were resuspended in 20 μL QUICKEXTRACT™ (DNA extraction buffer; Lucigen). Samples were then cycled in a PCR machine at 65° C. for 15 min, 68° C. for 15 min, 98° C. for 10 min. Samples were then frozen at −20° C. Samples were analyzed by NGS as described in Example 1.

FIG. 2 shows LDHA indels in HepG2 cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected within and/or adjacent to each of the LDHA target sites with each of the RNA guides. Therefore, LDHA-targeting RNA guides induced indels in exon 3 and exon 5 in HepG2 cells.

Example 3—Cas12i2-Mediated Editing of LDHA Target Sites in Primary Hepatocytes

This Example describes the genomic editing of the LDHA using Cas12i2 introduced into primary hepatocytes cells by RNP.

RNP complexation reactions were performed as described in Example 1 with RNA guides of Table 7. Primary hepatocyte cells from human donors were thawed from liquid nitrogen very quickly in a 37° C. water bath. The cells were added to pre-warmed hepatocyte recovery media (Thermofisher, CM7000) and centrifuged at 100 g for 10 minutes. The cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams' Medium E, Thermofisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum-containing), Thermofisher CM3000). The cells were subjected to trypan blue viability count with an INCUCYTE® disposable hemocytometer (Fisher scientific, 22-600-100). The cells were then washed in PBS and resuspended in P3 buffer+supplement (P3 PRIMARY CELL 4D-NUCLEOFECTOR™ X Kit; Lonza, VXP-3032) at a concentration of ˜7,500 cells/μL. Resuspended cells were dispensed at 150,000 cells/reaction into the 16 well Lonza NUCLEOCUVETTE strips or 500,000 cells/reaction into the single Lonza NUCLEOCUVETTES® for the mRNA readout. Complexed Cas12i2 RNP was added to each reaction at a final concentration of 20 μM (Cas12i2), and transfection enhancer oligos were then added at a final concentration of 4 μM. The final volume of each electroporated reaction was either 20 μL in the 16 well nucleocuvette strip format or 100 μL in the single nucleocuvette format. Non-targeting guides were used as negative controls.

The strips were electroporated using DS-150 program, while the single nucleocuvettes were electroporated using CA137 program (Lonza 4D-NUCLEOFECTOR™). Immediately following electroporation, pre-warmed Hepatocyte plating medium was added to each well and mixed very gently by pipetting. For each technical replicate plate, plated all the cell suspension of diluted nucleofected cells into a pre-warmed collagen-coated 96-well plate or 24-well plate (Thermofisher) with wells containing Hepatocyte plating medium. The cells were then incubated in a 37° C. incubator. The media was changed to hepatocyte maintenance media (Williams' Medium E, Thermofisher A1217601 supplemented with William's E medium Cell Maintenance Cocktail, Thermofisher CM 4000) after the cells attached after 4 hours. Fresh hepatocyte maintenance media was replaced after 2 days.

After 4-5 days post RNP electroporation, media was aspirated and the cells were harvested by shaking (500 rpm) in a 37° C. incubator with 2 mg/ml collagenase IV (Thermofisher, 17104019) dissolved in PBS containing Ca/Mg (Thermofisher). After cells were dissociated from the plate, they were transferred to 96-well TWIN.TEC® PCR plates (Eppendorf) and centrifuged. Media was flicked off and cell pellets for the NGS readout were resuspended in 20 μL. QUICKEXTRACT™ (DNA extraction buffer; Lucigen). Samples were then cycled in a PCR machine at 65° C. for 15 min, 68° C. for 15 min, 98° C. for 10 min and analyzed by NGS as described in Example 1.

For the mRNA readout, cell pellets were frozen at −80° C. and subsequently resuspended in lysis buffer and DNA/RNA extracted with the RNeasy kit (Qiagen) following manufacturer's instructions. The DNA extracted from the samples were analyzed by NGS. The RNA isolated was checked for quantity and purity using nanodrop, and subsequently used for cDNA synthesis using 5× iScript reverse transcription reaction mix (Bio-Rad laboratories), following manufacturer's recommendations. cDNA templated was appropriately diluted to be in linear range of the subsequent analysis. Diluted cDNA was used to set up a 20 μL Digital Droplet PCR (ddPCR-BioRad laboratories) reaction using target-specific primer and probe for LDHA, TTTTCCTTAGAACACCAAAGATTGTCTCTGGCAAAGACTATAATGTAACTGCAAAC TCCAAGCTGGTCATTATCACGGCTGGGGCACGTCAGCAAGAGGGAGAAAGCCGTC TTAATTTGGTCSEQ ID NO: 1264), and 2×ddPCR Supermix for Probes No dUTP (BioRad laboratories) following manufacturer's instructions. The reaction was used to generate droplets using Automated Droplet Generator (BioRad Laboratories), following manufacture's recommendations. The plate was sealed using PX1 PCR Plate Sealer (BioRad Laboratories) generated droplets were subjected to PCR amplification using C1000 Touch Thermal Cycler (BioRad Laboratories) using conditions recommended by the manufacturer. The PCR amplified droplets were read on QX200 Droplet Reader (BioRad Laboratories) and the acquired data was analyzed using QX Manager version 1.2 (BioRad Laboratories) to determine presence of absolute copy number of mRNA present in each reaction for the appropriate targets.

As shown in FIG. 3 , each RNA guide tested induced indels within and/or adjacent to the LDHA target sites. Indels were not induced with the non-targeting control. Therefore, LDHA-targeting RNA guides induced indels in primary hepatocytes. Indels for RNA guide E3T1 were then correlated with mRNA levels to determine whether indels led to mRNA knockdown and subsequent protein knockdown. FIG. 4 shows % mRNA knockdown of LDHA in edited cells compared to unedited control cells. RNA guide E3T1 resulted in knockdown of LDHA mRNA.

This Example thus shows that LDHA can be targeted by Cas12i2 RNPs in mammalian cells such as primary human hepatocytes.

Example 4—Editing of LDHA Target Sites in HepG2 Cells with Cas12i2 Variants

This Example describes indel assessment on LDHA target sites using variants introduced into HepG2 cells by transient transfection.

The Cas12i2 variants of SEQ ID NO: 1168 and SEQ ID NO: 1171 were individually cloned into a pcda3.1 backbone (Invitrogen). Nucleic acids encoding RNA guides E3T1, E3T3, EST1, E5T9, and E5T10 (Table 7) were cloned into a pUC19 backbone (New England Biolabs). The plasmids were then maxi-prepped and diluted.

HepG2 cells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; ThermoFisher) and counted. Cells were washed once with PBS and resuspended in SF buffer+supplement (SF CELL LINE 4D-NUCLEOFECTOR™ X KIT S; Lonza #V4XC-2032).

Approximately 16 hours prior to transfection, 25,000 HepG2 cells in EMEM/10% FBS were plated into each well of a 96-well plate. On the day of transfection, the cells were 70-90% confluent. For each well to be transfected, a mixture of Lipofectamine™ 3000 and Opti-MEM® was prepared and then incubated at room temperature for 5 minutes (Solution 1). After incubation, the Lipofectamine™:OptiMEM® mixture was added to a separate mixture containing nuclease plasmid and RNA guide plasmid and P3000 reagent (Solution 2). In the case of negative controls, the crRNA was not included in Solution 2. The Solution 1 and Solution 2 were mixed by pipetting up and down and then incubated at room temperature for 15 minutes. Following incubation, the Solution 1 and Solution 2 mixture was added dropwise to each well of a 96 well plate containing the cells.

After 3 days, wells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; ThermoFisher) and transferred to 96-well TWIN.TEC® PCR plates (Eppendorf). Media was flicked off and cells were resuspended in 20 μL QUICKEXTRACT™ (DNA extraction buffer; Lucigen). Samples were then cycled in a PCR machine at 65° C. for 15 min, 68° C. for 15 min, 98° C. for 10 min. Samples were then frozen at −20° C. and analyzed by NGS as described in Example 1.

As shown in FIG. 5A, two guides, E3T3 and E5T1, demonstrated significantly higher activity with variant Cas12i2 of SEQ ID NO: 1171 compared to variant Cas12i2 of SEQ ID NO: 1168. Comparable indel activity with the two Cas12i2 variants was observed for E3T1, E5T9, and E5T10. FIG. 5B shows the indel size frequency (left) and indel start position relative to the PAM for E5T9 and the variant Cas12i2 of SEQ ID NO: 1168 in HepG2 cells. As shown on the left, deletions ranged in size from 1 nucleotide to about 40 nucleotides. The majority of the deletions were about 8 nucleotides to about 23 nucleotides in length. As shown on the right, the target sequence is represented as starting at position 0 and ending at position 20. Indels started within about 5 nucleotides and about 35 nucleotides downstream of the PAM sequence. The majority of indels started about 10 nucleotides to about 30 nucleotides downstream of the PAM sequence.

Thus, this Example shows that LDHA is capable of being targeted by multiple Cas12i2 polypeptides.

Example 5—Editing of LDHA in Primary Human Hepatocytes Using Cas12i2 mRNA Constructs

This Example describes indel assessment on LDHA target sites via delivery of Cas12i2 mRNA and chemically modified LDHA-targeting RNA guides. mRNA sequences corresponding to the variant Cas12i2 sequence of SEQ ID NO: 1168 and the variant Cas12i2 sequence of SEQ ID NO: 1171 were synthesized by Aldeveron with 1-pseudo-U modified nucleotides and using CleanCap® Reagent AG (TriLink Biotechnologies). The Cas12i2 mRNA sequences, shown in Table 8, further comprised a C-terminal NLS.

TABLE 8 Cas12i2 mRNA Sequences Description mRNA sequence mRNA AUGAGCUCCGCCAUCAAGUCCUACAAGUCUGUGCUGCGGCCAAACGAGAGAAAGAAUCAGC corresponding to UGCUGAAGUCCACCAUCCAGUGCCUGGAGGACGGCUCCGCCUUCUUUUUCAAGAUGCUGCA variant Cas12i2 GGGCCUGUUUGGCGGCAUCACCCCCGAGAUCGUGAGAUUCAGCACAGAGCAGGAGAAGCAG of SEQ ID NO: CAGCAGGAUAUCGCCCUGUGGUGUGCCGUGAAUUGGUUCAGGCCUGUGAGCCAGGACUCCC 1168 UGACCCACACAAUCGCCUCCGAUAACCUGGUGGAGAAGUUUGAGGAGUACUAUGGCGGCAC AGCCAGCGACGCCAUCAAGCAGUACUUCAGCGCCUCCAUCGGCGAGUCCUACUAUUGGAAU GACUGCCGCCAGCAGUACUAUGAUCUGUGUCGGGAGCUGGGCGUGGAGGUGUCUGACCUGA CCCACGAUCUGGAGAUCCUGUGCCGGGAGAAGUGUCUGGCCGUGGCCACAGAGAGCAACCA GAACAAUUCUAUCAUCAGCGUGCUGUUUGGCACCGGCGAGAAGGAGGAUAGGUCUGUGAAG CUGCGCAUCACAAAGAAGAUCCUGGAGGCCAUCAGCAACCUGAAGGAGAUCCCAAAGAAUG UGGCCCCCAUCCAGGAGAUCAUCCUGAAUGUGGCCAAGGCCACCAAGGAGACAUUCAGACA GGUGUACGCAGGAAACCUGGGAGCACCAUCCACCCUGGAGAAGUUUAUCGCCAAGGACGGC CAGAAGGAGUUCGAUCUGAAGAAGCUGCAGACAGACCUGAAGAAAGUGAUCCGGGGCAAGU CUAAGGAGAGAGAUUGGUGCUGUCAGGAGGAGCUGAGGAGCUACGUGGAGCAGAAUACCAU CCAGUAUGACCUGUGGGCCUGGGGCGAGAUGUUCAACAAGGCCCACACCGCCCUGAAGAUC AAGUCCACAAGAAACUACAAUUUUGCCAAGCAGAGGCUGGAGCAGUUCAAGGAGAUCCAGU CUCUGAACAAUCUGCUGGUGGUGAAGAAGCUGAACGACUUUUUCGAUAGCGAGUUUUUCUC CGGCGAGGAGACCUACACAAUCUGCGUGCACCACCUGGGCGGCAAGGACCUGUCCAAGCUG UAUAAGGCCUGGGAGGACGAUCCCGCCGAUCCUGAGAAUGCCAUCGUGGUGCUGUGCGACG AUCUGAAGAACAAUUUUAAGAAGGAGCCUAUCAGGAACAUCCUGCGCUACAUCUUCACCAU CCGCCAGGAGUGUAGCGCACAGGACAUCCUGGCAGCAGCAAAGUACAAUCAGCAGCUGGAU CGGUAUAAGAGCCAGAAGGCCAACCCAUCCGUGCUGGGCAAUCAGGGCUUUACCUGGACAA ACGCCGUGAUCCUGCCAGAGAAGGCCCAGCGGAACGACAGACCCAAUUCUCUGGAUCUGCG CAUCUGGCUGUACCUGAAGCUGCGGCACCCUGACGGCAGAUGGAAGAAGCACCACAUCCCA UUCUACGAUACCCGGUUUUUCCAGGAGAUCUAUGCCGCCGGCAAUAGCCCUGUGGACACCU GUCAGUUUAGGACACCCCGCUUCGGCUAUCACCUGCCUAAGCUGACCGAUCAGACAGCCAU CCGCGUGAACAAGAAGCACGUGAAGGCAGCAAAGACCGAGGCACGGAUCAGACUGGCCAUC CAGCAGGGCACACUGCCAGUGUCCAAUCUGAAGAUCACCGAGAUCUCCGCCACAAUCAACU CUAAGGGCCAGGUGCGCAUCCCCGUGAAGUUUCGGGUGGGAAGGCAGAAGGGAACCCUGCA GAUCGGCGACCGGUUCUGCGGCUACGAUCAGAACCAGACAGCCUCUCACGCCUAUAGCCUG UGGGAGGUGGUGAAGGAGGGCCAGUACCACAAGGAGCUGGGCUGUUUUGUGCGCUUCAUCU CUAGCGGCGACAUCGUGUCCAUCACCGAGAACCGGGGCAAUCAGUUUGAUCAGCUGUCUUA UGAGGGCCUGGCCUACCCCCAGUAUGCCGACUGGAGAAAGAAGGCCUCCAAGUUCGUGUCU CUGUGGCAGAUCACCAAGAAGAACAAGAAGAAGGAGAUCGUGACAGUGGAGGCCAAGGAGA AGUUUGACGCCAUCUGCAAGUACCAGCCUAGGCUGUAUAAGUUCAACAAGGAGUACGCCUA UCUGCUGCGGGAUAUCGUGAGAGGCAAGAGCCUGGUGGAGCUGCAGCAGAUCAGGCAGGAG AUCUUUCGCUUCAUCGAGCAGGACUGUGGAGUGACCCGCCUGGGAUCUCUGAGCCUGUCCA CCCUGGAGACAGUGAAGGCCGUGAAGGGCAUCAUCUACUCCUAUUUUUCUACAGCCCUGAA UGCCUCUAAGAACAAUCCCAUCAGCGACGAGCAGCGGAAGGAGUUUGAUCCUGAGCUGUUC GCCCUGCUGGAGAAGCUGGAGCUGAUCAGGACUCGGAAGAAGAAGCAGAAGGUGGAGAGAA UCGCCAAUAGCCUGAUCCAGACAUGCCUGGAGAACAAUAUCAAGUUCAUCAGGGGCGAGGG CGACCUGUCCACCACAAACAAUGCCACCAAGAAGAAGGCCAACUCUAGGAGCAUGGAUUGG CUGGCCAGAGGCGUGUUUAAUAAGAUCCGGCAGCUGGCCCCAAUGCACAACAUCACCCUGU UCGGCUGCGGCAGCCUGUACACAUCCCACCAGGACCCUCUGGUGCACAGAAACCCAGAUAA GGCCAUGAAGUGUAGAUGGGCAGCAAUCCCAGUGAAGGACAUCGGCGAUUGGGUGCUGAGA AAGCUGUCCCAGAACCUGAGGGCCAAGAAUCGGGGCACCGGCGAGUACUAUCACCAGGGCG UGAAGGAGUUCCUGUCUCACUAUGAGCUGCAGGACCUGGAGGAGGAGCUGCUGAAGUGGCG GUCUGAUAGAAAGAGCAACAUCCCUUGCUGGGUGCUGCAGAAUAGACUGGCCGAGAAGCUG GGCAACAAGGAGGCCGUGGUGUACAUCCCAGUGAGGGGCGGCCGCAUCUAUUUUGCAACCC ACAAGGUGGCAACAGGAGCCGUGAGCAUCGUGUUCGACCAGAAGCAAGUGUGGGUGUGUAA UGCAGAUCACGUGGCAGCAGCAAACAUCGCACUGACCGGCAAGGGCAUCGGCGAGCAGUCC UCUGACGAGGAGAACCCCGAUGGCUCCAGGAUCAAGCUGCAGCUGACAUCUAAAAGGCCGG CGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGUAA (SEQ ID NO: 1265) mRNA AUGAGCUCCGCCAUCAAGUCCUACAAGUCUGUGCUGCGGCCAAACGAGAGAAAGAAUCAGC corresponding to UGCUGAAGUCCACCAUCCAGUGCCUGGAGGACGGCUCCGCCUUCUUUUUCAAGAUGCUGCA variant Cas12i2 GGGCCUGUUUGGCGGCAUCACCCCCGAGAUCGUGAGAUUCAGCACAGAGCAGGAGAAGCAG of SEQ ID NO: CAGCAGGAUAUCGCCCUGUGGUGUGCCGUGAAUUGGUUCAGGCCUGUGAGCCAGGACUCCC 1171 UGACCCACACAAUCGCCUCCGAUAACCUGGUGGAGAAGUUUGAGGAGUACUAUGGCGGCAC AGCCAGCGACGCCAUCAAGCAGUACUUCAGCGCCUCCAUCGGCGAGUCCUACUAUUGGAAU GACUGCCGCCAGCAGUACUAUGAUCUGUGUCGGGAGCUGGGCGUGGAGGUGUCUGACCUGA CCCACGAUCUGGAGAUCCUGUGCCGGGAGAAGUGUCUGGCCGUGGCCACAGAGAGCAACCA GAACAAUUCUAUCAUCAGCGUGCUGUUUGGCACCGGCGAGAAGGAGGAUAGGUCUGUGAAG CUGCGCAUCACAAAGAAGAUCCUGGAGGCCAUCAGCAACCUGAAGGAGAUCCCAAAGAAUG UGGCCCCCAUCCAGGAGAUCAUCCUGAAUGUGGCCAAGGCCACCAAGGAGACAUUCAGACA GGUGUACGCAGGAAACCUGGGAGCACCAUCCACCCUGGAGAAGUUUAUCGCCAAGGACGGC CAGAAGGAGUUCGAUCUGAAGAAGCUGCAGACAGACCUGAAGAAAGUGAUCCGGGGCAAGU CUAAGGAGAGAGAUUGGUGCUGUCAGGAGGAGCUGAGGAGCUACGUGGAGCAGAAUACCAU CCAGUAUGACCUGUGGGCCUGGGGCGAGAUGUUCAACAAGGCCCACACCGCCCUGAAGAUC AAGUCCACAAGAAACUACAAUUUUGCCAAGCAGAGGCUGGAGCAGUUCAAGGAGAUCCAGU CUCUGAACAAUCUGCUGGUGGUGAAGAAGCUGAACGACUUUUUCGAUAGCGAGUUUUUCUC CGGCGAGGAGACCUACACAAUCUGCGUGCACCACCUGGGCGGCAAGGACCUGUCCAAGCUG UAUAAGGCCUGGGAGGACGAUCCCGCCGAUCCUGAGAAUGCCAUCGUGGUGCUGUGCGACG AUCUGAAGAACAAUUUUAAGAAGGAGCCUAUCAGGAACAUCCUGCGCUACAUCUUCACCAU CCGCCAGGAGUGUAGCGCACAGGACAUCCUGGCAGCAGCAAAGUACAAUCAGCAGCUGGAU CGGUAUAAGAGCCAGAAGGCCAACCCAUCCGUGCUGGGCAAUCAGGGCUUUACCUGGACAA ACGCCGUGAUCCUGCCAGAGAAGGCCCAGCGGAACGACAGACCCAAUUCUCUGGAUCUGCG CAUCUGGCUGUACCUGAAGCUGCGGCACCCUGACGGCAGAUGGAAGAAGCACCACAUCCCA UUCUACGAUACCCGGUUUUUCCAGGAGAUCUAUGCCGCCGGCAAUAGCCCUGUGGACACCU GUCAGUUUAGGACACCCCGCUUCGGCUAUCACCUGCCUAAGCUGACCGAUCAGACAGCCAU CCGCGUGAACAAGAAGCACGUGAAGGCAGCAAAGACCGAGGCACGGAUCAGACUGGCCAUC CAGCAGGGCACACUGCCAGUGUCCAAUCUGAAGAUCACCGAGAUCUCCGCCACAAUCAACU CUAAGGGCCAGGUGCGCAUCCCCGUGAAGUUUCGGGUGGGAAGGCAGAAGGGAACCCUGCA GAUCGGCGACCGGUUCUGCGGCUACGAUCAGAACCAGACAGCCUCUCACGCCUAUAGCCUG UGGGAGGUGGUGAAGGAGGGCCAGUACCACAAGGAGCUGCGGUGUCGGGUGCGCUUCAUCU CUAGCGGCGACAUCGUGUCCAUCACCGAGAACCGGGGCAAUCAGUUUGAUCAGCUGUCUUA UGAGGGCCUGGCCUACCCCCAGUAUGCCGACUGGAGAAAGAAGGCCUCCAAGUUCGUGUCU CUGUGGCAGAUCACCAAGAAGAACAAGAAGAAGGAGAUCGUGACAGUGGAGGCCAAGGAGA AGUUUGACGCCAUCUGCAAGUACCAGCCUAGGCUGUAUAAGUUCAACAAGGAGUACGCCUA UCUGCUGCGGGAUAUCGUGAGAGGCAAGAGCCUGGUGGAGCUGCAGCAGAUCAGGCAGGAG AUCUUUCGCUUCAUCGAGCAGGACUGUGGAGUGACCCGCCUGGGAUCUCUGAGCCUGUCCA CCCUGGAGACAGUGAAGGCCGUGAAGGGCAUCAUCUACUCCUAUUUUUCUACAGCCCUGAA UGCCUCUAAGAACAAUCCCAUCAGCGACGAGCAGCGGAAGGAGUUUGAUCCUGAGCUGUUC GCCCUGCUGGAGAAGCUGGAGCUGAUCAGGACUCGGAAGAAGAAGCAGAAGGUGGAGAGAA UCGCCAAUAGCCUGAUCCAGACAUGCCUGGAGAACAAUAUCAAGUUCAUCAGGGGCGAGGG CGACCUGUCCACCACAAACAAUGCCACCAAGAAGAAGGCCAACUCUAGGAGCAUGGAUUGG CUGGCCAGAGGCGUGUUUAAUAAGAUCCGGCAGCUGGCCACCAUGCACAACAUCACCCUGU UCGGCUGCGGCAGCCUGUACACAUCCCACCAGGACCCUCUGGUGCACAGAAACCCAGAUAA GGCCAUGAAGUGUAGAUGGGCAGCAAUCCCAGUGAAGGACAUCGGCGAUUGGGUGCUGAGA AAGCUGUCCCAGAACCUGAGGGCCAAGAAUCGGGGCACCGGCGAGUACUAUCACCAGGGCG UGAAGGAGUUCCUGUCUCACUAUGAGCUGCAGGACCUGGAGGAGGAGCUGCUGAAGUGGCG GUCUGAUAGAAAGAGCAACAUCCCUUGCUGGGUGCUGCAGAAUAGACUGGCCGAGAAGCUG GGCAACAAGGAGGCCGUGGUGUACAUCCCAGUGAGGGGCGGCCGCAUCUAUUUUGCAACCC ACAAGGUGGCAACAGGAGCCGUGAGCAUCGUGUUCGACCAGAAGCAAGUGUGGGUGUGUAA UGCAGAUCACGUGGCAGCAGCAAACAUCGCACUGACCGGCAAGGGCAUCGGCCGGCAGUCC UCUGACGAGGAGAACCCCGAUGGCGGCAGGAUCAAGCUGCAGCUGACAUCUAAAAGGCCGG CGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGUAA (SEQ ID NO: 1266)

Cas12i2 RNA guides were designed and ordered from Integrated DNA Technologies (IDT) as having 3′ end modified phosphorothioated 2′ O-methyl bases or 5′ end and 3′ end modified phosphorothioated 2′ O-methyl bases guides, as specified in Table 9. Each variant Cas12i2 mRNA was mixed with a crRNA at a 1:1 (Cas12i2:crRNA) volume ratio (1050:1 crRNA:Cas12i2 molar ratio). The mRNA and crRNA were mixed immediately before electroporation. The primary human hepatocyte cells were cultured and electroporated as described in Example 3.

TABLE 9 Chemically Modified RNA Guide Sequences RNA Guide Sequence 3′ end modified AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGA*mA*mC* E3T1 mU (SEQ ID NO: 1267) 5′ and 3′ end mA*mG*mA*AAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGA* modified E3T1 mA*mC*mU (SEQ ID NO: 1268)

FIG. 6 shows editing of an LDHA target site by a variant Cas12i2 mRNA and 3′ end modified E3T1 (SEQ ID NO: 1267) or 5′ and 3′ end modified E3T1 (SEQ ID NO: 1268) RNA guide. Indels in the LDHA target site were introduced following electroporation of the Cas12i2 mRNA of SEQ ID NO: 1265 or SEQ ID NO: 1266 and either the RNA guide of SEQ ID NO: 1267 or SEQ ID NO: 1268. A higher percentage of NGS reads exhibited indels for RNA guide E3T1 with 5′ and 3′ end modifications (SEQ ID NO: 1268) compared to NGS reads for RNA guide with 3′ end modifications only (SEQ ID NO: 1267). Approximately 50% of NGS reads comprised indels following electroporation of the Cas12i2 mRNA of SEQ ID NO: 1266 and the RNA guide of SEQ ID NO: 1268.

This Example thus shows that LDHA can be targeted by Cas12i2 mRNA constructs and chemically modified RNA guides in mammalian cells.

Example 6—Off-Target Analysis of Cas12i2 and LDHA-Targeting RNA Guides

This Example describes on-target versus off-target assessment of a Cas12i2 variant and an LDHA-targeting RNA guide.

HEK293T cells were transfected with a plasmid encoding the variant Cas12i2 of SEQ ID NO: 1168 or the variant Cas12i2 of SEQ ID NO: 1171 and a plasmid encoding E3T1 (SEQ ID NO: 1214), EST1 (SEQ ID NO: 1221), E5T9 (SEQ ID NO: 1224), or E5T10 (SEQ ID NO: 1225) according the method described in Example 16 of PCT/US21/25257. The tagmentation-based tag integration site sequencing (TTISS) method described in Example 16 of PCT/US21/25257 was then carried out.

FIG. 7A and FIG. 7B show plots depicting on-target and off-target TTISS reads. The black wedge and centered number represent the fraction of on-target TTISS reads. Each grey wedge represents a unique off-target site identified by TTISS. The size of each grey wedge represents the fraction of TTISS reads mapping to a given off-target site. FIG. 7A shows TTISS reads for variant Cas12i2 of SEQ ID NO: 1168, and FIG. 7B shows TTISS reads for variant Cas12i2 of SEQ ID NO: 1171.

As shown in FIG. 7A, variant Cas12i2 of SEQ ID NO: 1168 paired with E5T9 demonstrated a low likelihood of off-target editing, as 100% of TTISS reads mapped to the on-target. No TTISS reads mapped to potential off-target sites. E3T1 and E5T10 also showed a low likelihood of off-target editing. For E3T1, 98% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 2% of TTISS reads. For E5T10, 97% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 3% of TTISS reads. E5T1 demonstrated a higher likelihood of off-target editing using the TTISS method.

As shown in FIG. 7B, variant Cas12i2 of SEQ ID NO: 1171 paired with the E5T9 demonstrated a low likelihood of off-target editing, as 100% of TTISS reads in replicate 1 and 93% of TTISS reads in replicate 2 mapped to the on-target, and two potential off-target sites represented the remaining 7% of TTISS reads in replicate 2. E5T10 also showed a low likelihood of off-target editing; 92% of TTISS reads in replicate 1 and 100% of TTISS reads in replicate 2 mapped to the on-target, and two potential off-target sites represented the remaining 8% of TTISS reads in replicate 1. Variant Cas12i2 of SEQ ID NO: 1171 paired with the E3T1 demonstrated a higher likelihood of off-target editing. 86% and 93% of TTISS reads mapping to the on-target in replicate 1 and replicate 2, respectively. 5 potential off-target sites represented the remaining 14% of TTISS reads in replicate 1, and 2 potential off-target sites represented the remaining 7% off TTISS reads in replicate 2 for E3T1.

Therefore, this Example shows that compositions comprising Cas12i2 and LDHA-targeting RNA guides comprise different off-target activity profiles.

Example 7—LDHA Protein Knockdown with Cas12i2 and LDHA-Targeting RNA Guides

This Example describes use of a Western Blot to identify knockdown of LDHA protein using variant Cas12i2 of SEQ ID NO: 1168 and LDHA-targeting RNA guides.

Primary hepatocyte cells from human donors were thawed from liquid nitrogen very quickly in a 37° C. water bath. The cells were added to pre-warmed hepatocyte recovery media (Thermo Fisher, CM7000) and centrifuged at 100 g for 10 minutes. The cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams' Medium E, Thermo Fisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum-containing), Thermo Fisher CM3000). The cells were subjected to trypan blue viability count with an Inucyte disposable hemocytometer (Fisher scientific, 22-600-100). The cells were then washed in PBS and resuspended in P3 buffer+supplement (Lonza, VXP-3032) at a concentration of ˜5000 cells/μL. Resuspended cells were dispensed at 500,000 cells/reaction into Lonza electroporation cuvettes

For the RNP reactions, E3T1 (SEQ ID NO: 1214), E5T9 (SEQ ID NO: 1224), E5T1 (SEQ ID NO: 1221), and E5T10 (SEQ ID NO: 1225) were used as the LDHA-targeting RNA guides. RNPs were added to each reaction at a final concentration of 20 μM (Cas12i2), and transfection enhancer oligos were then added at a final concentration of 4 Unelectroporated cells and cells electroporated without cargo were used as negative controls.

The strips were electroporated using an electroporation device (program CA137, Lonza 4D-nucleofector). Immediately following electroporation, pre-warmed Hepatocyte plating medium was added to each well and mixed very gently by pipetting. For each technical replicate plate, 500,000 cells of diluted nucleofected cells were plated into a pre-warmed collagen-coated 24-well plate (Thermo Fisher) with wells containing Hepatocyte plating medium. The cells were then incubated at 37° C. The media was changed to hepatocyte maintenance media (Williams' Medium E, Thermo Fisher A1217601 supplemented with William's E medium Cell Maintenance Cocktail, Thermo Fisher CM 4000) after the cells attached after 24 hours. Fresh hepatocyte maintenance media was replaced every 48 hours.

7 days post RNP electroporation, the media was aspirated, and the cells were washed gently with PBS. Cells were then lysed with RIPA Lysis and Extraction buffer (Thermo Fisher 89901)+1× protease inhibitors (Thermo Fisher 78440) for 30 minutes on ice, mixing the samples every 5 minutes. Cell lysate was quantified via Pierce BCA Protein Assay Kit (Thermo Fisher 23227). 15 μg of total protein per sample was prepared for SDS-PAGE in 1× Laemmlli Sample buffer (BioRad 1610747) and 100 mM DTT, then heated at 95° C. for 10 minutes. Samples were run on a 4-15% TGX gel (BioRad 5671084) at 200V for 45 minutes. Samples were transferred to a 0.2 um nitrocellulose membrane (BioRad 1704159) using the Trans Blot Turbo System. The membrane was blocked in Intercept TBS Blocking Buffer (Li-cor 927-60001) for 30 minutes at room temperature. The blot was then incubated in a 1:1000 dilution of primary anti-LDHA antibody (Abcam ab52488) and 1:2500 dilution of primary anti-vinculin antibody (Sigma V9131) in blocking buffer at 4 C overnight. The blot was washed three times with TBST (Thermo Fisher 28360) for 5 minutes each, then incubated with a 1:12500 dilution of IR680 anti-mouse (Thermo Fisher PI35518) and IR800 anti-rabbit secondary antibodies (Thermo Fisher PISA535571) in TBST for 1 hour at room temperature. The blot was then washed three times with TBST for 5 minutes each and visualized on the Li-cor Odyssey CLX.

Knockdown of LDHA protein (monomer and dimer) was observed in primary human hepatocytes at Day 7 post editing by Cas12i2 RNPs targeting the LDHA gene (FIG. 8 ). This knockdown was seen across each of the four RNA guides, E3T1, E5T9, EST1, and E5T10 (lanes 1-8). LDHA knockdown was not observed for the buffer only (lanes 9 and 10) or unelectroporated controls (lanes 11 and 12).

This Example thus shows that LDHA protein levels were decreased following editing with Cas12i2 and LDHA-targeting RNA guides.

Other Embodiments

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. 

1. A gene editing system for genetic editing of a lactate dehydrogenase A (LDHA) gene, comprising (i) a Cas12i2 polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide, wherein the Cas12i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1166 and comprises one or more mutations relative to SEQ ID NO: 1166; (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence.
 2. The gene editing system of claim 1, wherein the one or more mutations in the Cas12i2 polypeptide are at positions D581, G624, F626, P868, I926, V1030, E1035, and/or S1046 of SEQ ID NO:
 1166. 3. The gene editing system of claim 2, wherein the one or more mutations are amino acid substitutions, which optionally is D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.
 4. The gene editing system of claim 3, wherein the Cas12i2 polypeptide comprises: (i) mutations at positions D581, D911, I926, and V1030, which optionally are amino acid substitutions of D581R, D911R, I926R, and V1030G; (ii) mutations at positions D581, I926, and V1030, which optionally are amino acid substitutions of D581R, I926R, and V1030G; (iii) mutations at positions D581, I926, V1030, and S1046, which optionally are amino acid substitutions of D581R, I926R, V1030G, and S1046G; (iv) mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046, which optionally are amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G; or (v) mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046, which optionally are amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G.
 5. The gene editing system of claim 1, wherein the Cas12i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 1167, 1168, 1169, 1170, or
 1171. 6. The gene editing system of claim 1, which comprises the first nucleic acid encoding the Cas12i2 polypeptide.
 7. The gene editing system of claim 6, wherein the first nucleic acid is a messenger RNA (mRNA), and/or is included in a viral vector.
 8. (canceled)
 9. The gene editing system of claim 1, wherein the target sequence is within exon 3 or exon 5 of the LDHA gene, and/or comprises: (i) (SEQ ID NO: 1237) 5′-TAGGACTTGGCAGATGAACT-3′; (ii) (SEQ ID NO: 1239) 5′-GATGACATCAACAAGAGCAA-3; (iii) (SEQ ID NO: 1245) 5′-TTCATAGTGGATATCTTGAC-3′; (iv) (SEQ ID NO: 1248) 5′-TCATAGTGGATATCTTGACC-3′; or (v) (SEQ ID NO: 1249) 5′-CATAGTGGATATCTTGACCT-3′.


10. (canceled)
 11. The gene editing system of claim 9, wherein the spacer sequence comprises: (i) (SEQ ID NO: 1269) 5′-UAGGACUUGGCAGAUGAACU-3′; (ii) (SEQ ID NO: 1270) 5′-GAUGACAUCAACAAGAGCAA-3′; (iii) (SEQ ID NO: 1271) 5′-UUCAUAGUGGAUAUCUUGAC-3′; (iv) (SEQ ID NO: 1272) 5′-UCAUAGUGGAUAUCUUGACC-3′; or (v)  (SEQ ID NO: 1273) 5′-CAUAGUGGAUAUCUUGACCU-3.


12. (canceled)
 13. The gene editing system of claim 1, wherein the RNA guide comprises the spacer sequence and a direct repeat sequence, which is at least 90% identical to any one of SEQ ID NOs: 1-10 or a fragment thereof that is at least 23-nucleotide in length. 14-16. (canceled)
 17. The gene editing system of claim 13, wherein the direct repeat sequence is 5′-AGAAAUCCGUCUUUCAUUGACGG-3′ (SEQ ID NO: 10).
 18. The gene editing system of claim 1, wherein the RNA guide comprises the nucleotide sequence of: (i) (SEQ ID NO: 1214) 5′-AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGAACU-3′; (ii) (SEQ ID NO: 1235) 5′-AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGCAA-3′; (iii) (SEQ ID NO: 1221) 5′-AGAAAUCCGUCUUUCAUUGACGGUUCAUAGUGGAUAUCUUGAC-3′; (iv) (SEQ ID NO: 1224) 5′-AGAAAUCCGUCUUUCAUUGACGGUCAUAGUGGAUAUCUUGACC-3′; or (v) (SEQ ID NO: 1225) 5′-AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU-3′.


19. The gene editing system of claim 1, wherein the system comprises the second nucleic acid encoding the RNA guide, or wherein the nucleic acid encoding the RNA guide is located in a viral vector.
 20. (canceled)
 21. The gene editing system of claim 7, wherein the viral vector comprises both the first nucleic acid encoding the Cas12i2 polypeptide and the second nucleic acid encoding the RNA guide.
 22. The gene editing system of claim 21, wherein the system comprises the first nucleic acid encoding the Cas12i2 polypeptide, which is located on a first vector, and wherein the system comprises the second nucleic acid encoding the RNA guide, which is located on a second vector; or wherein the system comprises one or more lipid nanoparticles (LNPs), which encompass (i), (ii), or both. 23-24. (canceled)
 25. The gene editing system of claim 22, wherein the system comprises the LNP, which encompass (i), and wherein the system comprises a viral vector comprising the second nucleic acid encoding the RNA guide; or wherein the system comprises the LNP, which encompass (ii), and wherein the system comprises a viral vector comprising the first nucleic acid encoding Cas12i2 polypeptide.
 26. The gene editing system of claim 25, wherein the viral vector is an AAV vector.
 27. A gene editing system for genetic editing of a lactate dehydrogenase A (LDHA) gene, comprising (i) a Cas12i polypeptide or a first nucleic acid encoding the Cas12i polypeptide, optionally wherein the Cas12i polypeptide is a Cas12i2 polypeptide; and (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within exon 3 or exon 5 of an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence. 28-46. (canceled)
 47. A pharmaceutical composition comprising the gene editing system set forth in claim
 1. 48. A kit comprising the elements (i) and (ii) of the gene editing system set forth in claim
 1. 49. A method for editing a lactate dehydrogenase A (LDHA) gene in a cell, the method comprising contacting a host cell with the gene editing system for editing the LDHA gene set forth in claim 1 to genetically edit the LDHA gene in the host cell. 50-52. (canceled)
 53. A method for treating primary hyperoxaluria (PH) in a subject, comprising administering to a subject in need thereof a gene editing system for editing a lactate dehydrogenase A (LDHA) gene set forth in claim
 1. 54-55. (canceled)
 56. An RNA guide, comprising (i) a spacer sequence that is specific to a target sequence in a lactate dehydrogenase A (LDHA) gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence; and (ii) a direct repeat sequence. 57-65. (canceled) 